Cationic lipids involved in gene transfer mobilize intracellular calcium

Cationic lipids are efficient tools to introduce nucleic acids and proteins into cells. Elucidation of the mechanism and cellular pathways associated with such transport has been relatively tedious, even though significant progress has been made in the characterization of the intracellular trafficking of lipid/DNA complexes. Surprisingly little is known about the effects of these delivery vectors on cell functioning. In this report, we show that both cationic lipids and cationic lipid/DNA complexes mobilize the intracellular calcium. Removal of extracellular calcium did not significantly abolish this effect and preincubating cells with thapsigargin led to a decrease in [Ca²⁺]_i, indicating that calcium was released mainly from internal calcium stores sensitive to thapsigargin. Pretreatment of the cells with the phospholipase C inhibitor U73122, blocked the [Ca²⁺]_i rise, suggesting an inositol dependent mechanism.     

Cationic nucleoside lipids for gene delivery

A novel uridine-based nucleo-lipid, DOTAU (N-[5'-(2',3'-dioleoyl)uridine]-N',N',N'-trimethylammonium tosylate) was prepared by using a convenient four-step synthetic pathway. From the preliminary physicochemical studies (quasielastic light scattering and light microscopy), this amphiphilic structure forms supramolecular organizations in aqueous solution. In addition, in the presence of nucleic acids, transmission electronic microscopy experiments (TEM) and small angle X-ray scattering (SAXS) reveal the formation of multilamellar structures similar to lipoplexes (cationic liposome-DNA complexes) with cationic lipids. The formation of a complex was confirmed by fluorescence spectroscopic assays involving ethidium bromide. Transfection assays of mammalian cell lines (HeLa and MCF-7) indicate that DOTAU can transfect efficiently an expression vector (pEGFP) encoding GFP. Proliferation assays realized on these cell lines show that DOTAU does not inhibit cell proliferation and is less toxic than the commercial Lipofectamine 2000.     

阳离子脂质体与基因转移

阳离子脂质体是表达带正电的脂南体。它可与带负电的基因（ＤＮＡ或ＲＮＡ）形成带正电的脂－基因复合物,此复合物借静电作用吸附于细胞表面,通过与细胞膜融合或细胞内吞而将结合的基因导入细胞,并获得表达。阳离子脂质体转基因的操作简便、重复性好,杂效率高,在基因工程、物种培育和基因治疗研究等方面有广阔的应用前景。     

Cationic lipids with increased DNA binding affinity for nonviral gene transfer in dividing and nondividing cells

Effect of headgroup structure on catonic lipid-mediated transfection was investigated with either a (i) tertiary amine, (ii) quaternary amine with a hydroxyl, or (iii) quaternary amine with mesylate as headgroups. Liposomes were formulated using cholesterol or dioleoyl phosphatidyl ethanolamine (DOPE) as colipids, and transfection efficiencies were determined in rapidly dividing colon carcinoma (CT 26) and rat aortic smooth muscle (RASM) cells as well as in nondividing human pancreatic islets using luciferase and green fluorescent protein expression plasmids, pcDNA3-Luc and pCMS-EGFP, respectively. Liposome/pDNA complexes were evaluated for DNA conformational state by circular dichroism (CD), DNA condensation by electrophoretic mobility shift assay (EMSA), particle size and zeta potential by laser diffraction technique, and surface morphology by transmission electron microscopy (TEM). Encouraging transfection results were obtained with the mesylate headgroup based lipid in liposome formulations with DOPE as a colipid, which were higher than the commercially available Lipofectamine formulation. We hypothesize that the additional hydrogen bonding or covalent interactions of the headgroup with the plasmid DNA, leading to higher binding affinity of the cationic lipids to pDNA, results in higher transfection. This hypothesis is supported by TEM observations where elongated complexes were observed and more lipid was seen associated with the DNA.     

Cationic liposomal lipids: from gene carriers to cell signaling

Cationic lipids are positively charged amphiphilic molecules which, for most of them, form positively charged liposomes, sometimes in combination with a neutral helper lipid. Such liposomes are mainly used as efficient DNA, RNA or protein carriers for gene therapy or immunization trials. Over the past decade, significant progress has been made in the understanding of the cellular pathways and mechanisms involved in lipoplex-mediated gene transfection but the interaction of cationic lipids with cell components and the consequences of such an interaction on cell physiology remains poorly described. The data reported in the present review provide evidence that cationic lipids are not just carriers for molecular delivery into cells but do modify cellular pathways and stimulate immune or anti-inflammatory responses. Considering the wide number of cationic lipids currently available and the variety of cellular components that could be involved, it is likely that only a few cationic lipid-dependent functions have been identified so far.     

Sialic acid containing substrates as intracellular calcium receptors involved in transmitter release

The possible function of sialic acid containing substrates in the synaptic terminals was studied by intracellular injection of ruthenium red (RuR) and neuraminidase (NAA). When injected into cholinergic and non cholinergic neurons of Aplysia, NAA and RuR, known to have similar molecular targets, blocked synaptic transmission. The subcellular sites of action of these molecules were investigated. 1. ACh receptors are not affected by RuR. 2. An intracellular site of action of RuR is likely, as less was necessary to block transmission when injected into the presynaptic cell than when applied in the bath. 3. Ca++ channels are not blocked by RuR or neuraminidase. 4. Transmission block is not due to an axonal conduction block, since strong somatic depolarization is not able to induce transmitter release in the presence of RuR. 5. Biochemical analysis of pools of 3H ACh was performed in controls and after injection of RuR. RuR appeared to significantly increase the cytoplasmic ACh pool without any change of the vesicular ACh pool. 6. Quantal release of transmitter was analysed with a current fluctuation method. There were no changes in the amplitude or decay time (tau) of miniataure postsynaptic potentials, but a decrease in the quantal content of the synapse was found.     

Estrogen induces phospholipase A2 activation through ERK1/2 to mobilize intracellular calcium in MCF-7 cells

The principal secreted estrogen, 17beta-estradiol rapidly activates signaling cascades that regulate important physiological processes including ion transport across membranes, cytosolic pH and cell proliferation. These effects have been extensively studied in the MCF-7 estrogen-responsive human breast carcinoma cell line. Here, we demonstrate that a physiological concentration of 17beta-estradiol caused a rapid, synchronous and transient increase in intracellular calcium concentration in a confluent monolayer of MCF-7 cells 2-3 min after treatment. This response was abolished when cells were pre-incubated with the phospholipase A(2) (PLA(2)) inhibitor quinacrine or with the cyclooxygenase inhibitor indomethacin. The translocation of GFP-cPLA(2)alpha to perinuclear membranes occurred 1-2 min after 17beta-estradiol treatment; this translocation was concurrent with the transient phosphorylation of cPLA(2)alpha at serine residue 505. The phosphorylation and translocation of cPLA(2) were sensitive to inhibition of the extracellular signal regulated kinase (ERK) signaling cascade and occurred simultaneously with a transient activation of ERK. The phosphorylation of cPLA(2) could be stimulated by membrane impermeable 17beta-estradiol conjugated to bovine serum albumen and was blocked by an antagonist of the classical estrogen receptor. Here we show, for the first time, that PLA(2) and the eicosanoid biosynthetic pathway are involved in the 17beta-estradiol induced rapid calcium responses of breast cancer cells.     

Mechanisms involved in the increase in intracellular calcium following hypotonic shock in bovine articular chondrocytes

The extracellular osmotic environment of chondrocytes fluctuates during joint loading as fluid is expressed from and reimbibed by the extracellular matrix. Matrix synthesis by chondrocytes is modulated by joint loading, possibly mediated by variations in intracellular composition. The present study has employed the Ca2+-sensitive fluoroprobe Fura-2 to determine the effects of hypotonic shock (HTS) on intracellular Ca2+ concentration ([Ca2+]i) and to characterise the mechanisms involved in the response for isolated bovine articular chondrocytes. In cells subjected to a 50% dilution, [Ca2+]i rapidly increased by approximately 250%, a sustained plateau being achieved within 300 s. The effect was inhibited by thapsigargin or by removal of extracellular Ca2+, indicating that the rise in [Ca2+]i reflects both influx from the extracellular medium and release from intracellular stores. Inhibition of the response by neomycin implicates activation of PLC and IP3 synthesis in the mobilisation of Ca2+ from intracellular stores. The rise was insensitive to inhibitors of L-type voltage-activated Ca2+ channels (LVACC) or reverse mode Na+/Ca2+ exchange (NCE) but could be significantly attenuated by ruthenium red, an inhibitor of transient receptor potential vanilloid (TRPV) channels and by Gd3+, a blocker of stretch-activated cation (SAC) channels. The HTS-induced rise in [Ca2+]i was almost completely absent in cells treated with Ni2+, a non-specific inhibitor of Ca2+ entry pathways. We conclude that in response to HTS the opening of SACC and a member of TRPV channel family leads to Ca2+ influx, simultaneously with the release from intracellular stores.     

Lateral gene transfer in vitro in the intracellular pathogen Chlamydia trachomatis

Genetic recombinants that resulted from lateral gene transfer (LGT) have been detected in sexually transmitted disease isolates of Chlamydia trachomatis, but a mechanism for LGT in C. trachomatis has not been described. We describe here a system that readily detects C. trachomatis LGT in vitro and that may facilitate discovery of its mechanisms. Host cells were simultaneously infected in the absence of antibiotics with an ofloxacin-resistant mutant and a second mutant that was resistant to lincomycin, trimethoprim, or rifampin. Selection for doubly resistant C. trachomatis isolates in the progeny detected apparent recombinant frequencies of 10(-4) to 10(-3), approximately 10(4) times more frequent than doubly resistant spontaneous mutants in progeny from uniparental control infections. Polyclonal doubly resistant populations and clones isolated from them in the absence of antibiotics had the specific resistance-conferring mutations present in the parental mutants; absence of the corresponding normal nucleotides indicated that they had been replaced by homologous recombination. These results eliminate spontaneous mutation, between-strain complementation, and heterotypic resistance as general explanations of multiply resistant C. trachomatis that originated in mixed infections in our experiments and demonstrate genetic stability of the recombinants. The kind of LGT we observed might be useful for creating new strains for functional studies by creating new alleles or combinations of alleles of polymorphic loci and might also disseminate antibiotic resistance genes in vivo. The apparent absence of phages and conjugative plasmids in C. trachomatis suggests that the LGT may have occurred by means of natural DNA transformation. Therefore, the experimental system may have implications for genetically altering C. trachomatis by means of DNA transfer.     

Cationic lipids derived from glycine betaine promote efficient and non-toxic gene transfection in cultured hepatocytes

Background

The low efficiency and toxicity of transfection in a primary culture of hepatocytes using cationic lipids remains a limiting step to the study of gene function and the setting up of non‐viral gene therapy.

Methods

A novel class of cationic lipids (GBs) derived from natural glycine betaine compounds covalently linked to acyl chains by enzymatically hydrolysable peptide and ester bonds, a structure designed to reduce cytotoxicity, was used to improve transfection efficiency in a primary culture of rat hepatocytes. The relationship between lipid structure, lipoplex formulation and transfection efficiency was studied using six GBs (12‐14‐16, 22‐24‐26) varying in their spacer and acyl chains.

Results

GB12, characterized by short [(CH₂)₁₀] acyl chains and spacer, allowed plasmid uptake in all cells and reporter gene expression in up to 40% of hepatocytes with a low cytotoxicity, a much higher efficiency compared with transfections using other reagents including Fugene6^? and Lipofectin^?. We also showed that numerous cells accumulated high amounts of plasmids demonstrating that GB12 promoted a very efficient DNA transfer through plasma membrane leading to an increase in nuclear plasmid translocation, allowing a much higher gene expression. Moreover, GB12‐transfected hepatocytes survived to injection in normal livers and were found to express the LacZ reporter gene.

Conclusions

The non‐toxic GB12 formulation is a powerful vehicle for plasmid delivery in cultured hepatocytes with relevance in liver gene therapy. Copyright © 2002 John Wiley & Sons, Ltd.

     

The role of helper lipids in cationic liposome-mediated gene transfer.

In the procedure for cationic liposome-mediated transfection, the cationic lipid is usually mixed with a "helper lipid" to increase its transfection potency. The importance of helper lipids, including dioleoylphosphatidylcholine (DOPC) and phosphatidylethanolamine (dioleoyl PE), DO was examined. Freeze-fracture electron microscopy of DNA:cationic complexes containing the pSV-beta-GAL plasmid DNA, the cationic lipid dioleoyl trimethylammonium propane, and these helper lipids showed that the most efficient mixtures were aggregates of ensheathed DNA and fused liposomes. PE-containing complexes aggregated rapidly when added to culture media containing polyanions, whereas PC-containing complexes did not. However, more granules of PC-containing complexes were formed on cell surfaces after the complexes were added to Chinese hamster ovary (CHO) cells in transfection media. Pronase treatment inhibited transfection, whereas dilute poly-L-lysine enhanced transfection, indicating that the attachment of DNA:liposome complexes to cell surfaces was mediated by electrostatic interaction. Fluorescence spectroscopy studies confirmed that more PC-containing complexes than PE-containing complexes were associated with CHO cells, and that more PC-containing complexes were located in a low pH environment (likely to be within endosomes) with time. Cytochalasin-B had a stronger inhibitory effect on PC-containing liposome-mediated than on PE-containing liposome-mediated transfection. Confocal microscopic recording of the fluorescently label lipid and DNA uptake process indicated that many granules of DNA:cationic liposome complexes were internalized as a whole, whereas some DNA aggregates were left out on the cell surfaces after liposomes of the complexes fused with the plasma membranes. For CHO cells, endocytosis seems to be the main uptake pathway of DNA:cationic liposome complexes. More PC-containing granules than PE-containing granules were formed on cell surfaces by cytoskeleton-directed membrane motion, after their respective DNA:liposome complexes attached to cell surfaces by electrostatic means. Formation of granules on the cell surface facilitated and/or triggered endocytosis. Fusion between cationic liposomes and the cell membrane played a secondary role in determining transfection efficiency.     

Cationic lipids enhance siRNA-mediated interferon response in mice

RNA interference mediated by small interfering RNAs (siRNAs) shows promise as a powerful research tool for gene function studies. However, controversy exists over the potential of siRNA-induced interferon response in vitro and in vivo. In this study, we showed that although intravenous administration of siRNA alone is essentially inert, injection of siRNA complexed with cationic liposomes resulted in a potent induction of both type I and type II interferon responses. Furthermore, i.v. administration of cationic lipid/siRNA complexes led to activation of STAT1. This study suggests caution in data interpretation and the potential toxicity with in vivo use of siRNA, particularly when delivered via a cationic lipid vector. This study also suggests the potential of siRNA as an immunostimulatory agent for immunotherapy.     

Adenylyl cyclase pathway is involved in the regulation of intracellular calcium level regulation in brown preadipocytes

Mechanism of adrenergically activated calcium response in freshly isolated brown preadipocytes was studied with fluorescent probe Fura-2. Application of a direct activator of adenylylcyclase forskolin or cell permeable analog BrcAMP caused rise in the intracellular calcium level that was even higher than after the application of norepinephrine. Protein kinase A inhibitor H-89 in a dose-dependent manner reduced, while inhibitor of total phosphodiesterase activity IBMX, or protein phosphatase inhibitor ocadaic acid enhanced norepinephrine or isoproterenol initiated cellular calcium responses. It is concluded that cAMP and protein kinase A mediated phosphorylation play a crucial role in adrenergically initiated calcium signalling in brown preadipocytes.     

Cytosolic calcium is involved in the regulation of barley hemoglobin gene expression

Hemoglobin gene expression is upregulated during hypoxia. To determine whether the induction occurs via similar mechanisms that have been proposed for other hypoxically induced proteins, barley (Hordeum vulgare L.) aleurone layers were treated with various agents that interfere with known components of signal transduction. Ruthenium red, an organelle calcium channel blocker, inhibited anoxia-induced hemoglobin (Hb) and alcohol dehydrogenase (EC 1.1.1.1) (Adh) gene expression in a dose-dependent manner. The divalent ionophore, A23187, combined with EGTA also dramatically reduced anoxia-induced Hb and Adh expression. Normal induction of Hb by anoxia in EGTA-treated cells was restored by adding exogenous Ca²⁺ but not Mg²⁺, suggesting that cytosolic calcium is involved in Hb and Adh regulation. W-7, a calmodulin antagonist, did not affect anaerobically induced Hb and Adh expression even though it induced Hb under aerobiosis. A3, a protein kinase inhibitor, did not significantly affect anaerobically induced Hb, but did significantly upregulate the gene under aerobic conditions. The results indicate that calmodulin-independent anaerobic alteration in cytosolic Ca²⁺ and protein dephosphorylation are factors in Hb induction.     

Enhanced intracellular calcium induced by urocortin is involved in degranulation of rat lung mast cells.

Corticotropin-releasing factor (CRF), which activates the hypothalamic-pituitary- adrenal axis under stress, also has proinflammatory peripheral effects possibly through mast cells. The purpose of this study was to investigate the effect of urocortin (UCN), a 40-amino-acid CRF family peptide, on degranulation and intracellular calcium of rat lung mast cells. The activation and degranulation of mast cells were observed by Toluidine blue staining and transmission electron microscope. The intracellular calcium was investigated using confocal laser scanning microscopy and flow cytometry. The results indicated that all the three different concentrations of UCN (0.1, 1 and 10 microM) significantly induced the activation and degranulation of rat lung mast cells in vitro. This effect was markedly blocked by selective CRF receptor 1 (CRF-R1) antagonist antalarmin, but not by specific CRF receptor 2 (CRF-R2) antagonist antisauvagine-30 (anti-Svg-30). The results also showed that UCN caused a rapid peak increase in [Ca(2+)](i) at point of 300s after UCN treatment, followed by a decrease to a sustained plateau phase. The peak increase in [Ca(2+)](i) induced by UCN was significantly inhibited by antalarmin, but not by anti-Svg-30. This effect of UCN on [Ca(2+)](i) in rat lung mast cells was also found by flow cytometry. Regression analysis revealed a positive correlation between mast cells degranulation extent and the maximum value of [Ca(2+)](i) (P < 0.01). Taken together, our present study suggested that UCN induced the increase of [Ca(2+)](i) and degranulation of rat lung mast cells through CRF-R1. These findings may have implications for the pathophysiology of allergic and inflammatory lung disorders such as asthma, which is closely associated with mast cell activation and degranulation.     

Peptide-based intracellular shuttle able to facilitate gene transfer in mammalian cells.

Loligomers are peptide-based intracellular vehicles able to penetrate cells and self-localize into distinct cellular compartments. Loligomers can be rapidly assembled by automated solid-phase approaches and were designed to act as nonviral, nonlipophilic intracellular shuttles. One nucleus-directed loligomer, termed loligomer 4, was evaluated for its ability to function as a transfection agent. Loligomer 4 readily associates with plasmids to form noncovalent complexes. The migration of loligomer 4-plasmid complexes into cells was monitored by flow cytometry and fluorescence microscopy. Populations of plasmids labeled with 7-AAD exist either free or in association with loligomer 4 inside cells and are visible throughout the cytosol and nucleus of chinese hamster ovary (CHO) cells. Loligomer 4-plasmid complexes were not cytotoxic to cells and were readily imported by most cells (>70%). CHO cells were transfected with complexes of loligomer 4 and plasmids harboring luciferase, green fluorescent protein or beta-galactosidase reporter genes. The transfection efficiency of loligomer 4-plasmid DNA complexes was greater when cells were maintained as suspensions instead of monolayers. Transfections could be performed with cells suspended in serum-containing medium. The observed levels of transfection, however, were modest with 5-10% of CHO cells expressing either a green fluorescent protein or the enzyme beta-galactosidase. Loligomers have recently been observed in vesicular compartments [Singh, D., Kiarash, R., Kawamura, K, LaCasse, E. C., and Gariépy, J. (1998) Biochemistry 37, 5798-5809] and differences between levels of cellular import and transfection efficiency may well reflect the need to optimize the release of loligomers and their complexes from these compartments in future designs. In summary, loligomer 4 behaves as a stable, soluble and effective transfection agent. These results demonstrate the feasibility of designing loligomers able to act as intracellular guided agents aimed at gene transfer applications.     

Convective-diffusive mass transfer of agonist and the intracellular calcium response of endothelial cell

Endothelial cell layer regulates several crucial physiological processes of the vascular system. The mechanism of the response of this cell layer to the flow of surrounding fluid is still largely unclear. In the present article, a comparison of the available experimental results for the intracellular calcium ion concentration and theoretical results for the extra-cellular ATP concentration obtained using a convective-diffusive mass transfer model, has been made which supports the mass transfer model for the endothelial cell response to the fluid flow. The experimental results are in excellent agreement with the calculated values assuming the effect of ATP concentration alone.