Young K. Song

Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA

Development of methods that allow an efficient expression of exogenous genes in animals would provide tools for gene function studies, treatment of diseases and for obtaining gene products. Therefore, we have developed a hydrodynamics-based procedure for expressing transgenes in mice by systemic administration of plasmid DNA. Using cDNA of luciferase and β-galactosidase as a reporter gene, we demonstrated that an efficient gene transfer and expression can be achieved by a rapid injection of a large volume of DNA solution into animals via the tail vein. Among the organs expressing the transgene, the liver showed the highest level of gene expression. As high as 45 μg of luciferase protein per gram of liver can be achi- eved by a single tail vein injection of 5 μg of plasmid DNA into a mouse. Histochemical analysis using β-galactosidase gene as a reporter reveals that approximately 40% of hepatocytes express the transgene. The time–response curve shows that the level of transgene expression in the liver reaches the peak level in approximately 8 h after injection and decreases thereafter. The peak level of gene expression can be regained by repeated injection of plasmid DNA. These results suggest that a simple, convenient and efficient method has been developed and which can be used as an effective means for studying gene function, gene regulation and molecular pathophysiology through gene transfer, as well as for expressing proteins in animals.

Hydroporation as the mechanism of hydrodynamic delivery.

We have reported that a rapid tail vein injection of a large volume of plasmid DNA solution into a mouse results in high level of transgene expression in the liver. Gene transfer efficiency of this hydrodynamics-based procedure is determined by the combined effect of a large volume and high injection speed. Here, we show that the hydrodynamic injection induces a transient irregularity of heart function, a sharp increase in venous pressure, an enlargement of liver fenestrae, and enhancement of membrane permeability of the hepatocytes. At the cellular level, our results suggest that hepatic delivery by the hydrodynamic injection is accomplished by the generation of membrane pores in the hepatocytes.

Long-term expression of human alpha1-antitrypsin gene in mouse liver achieved by intravenous administration of plasmid DNA using a hydrodynamics-based procedure.

The liver is an important target organ for gene transfer due to its large capacity for synthesizing serum proteins and its involvement in numerous genetic and acquired diseases. Previously, we and others have shown that an efficient gene transfer to liver cells in vivo can be achieved by an intravenous injection of plasmid DNA using a hydrodynamics-based procedure. In this study, we systematically characterized the expression of transgene encoding a secretory protein in mouse. Using human α1-antitrypsin (hAAT) gene as a reporter, we demonstrate that the serum level of hAAT can reach as high as 0.5 mg/ml by a simple tail vein injection of 10–50 μg plasmid DNA into a mouse. The serum hAAT reaches the peak level 1 day after DNA injection and then declines during the following 2 to 4 weeks to 2–5 μg/ml, a level which persists for at least 6 months. Southern analysis of extracted DNA and RT-PCR analysis of RNA from the liver reveal that hAAT gene is active and present as episomal form after 6 months. These results suggest that the hydrodynamics-based transfection procedure provides a valuable tool for screening genes for therapeutic purposes in whole animals.

Recognition and clearance of liposomes containing phosphatidylserine are mediated by serum opsonin

Liver uptake of liposomes containing phosphatidylserine was studied in a single pass liver perfusion system and found to be serum dependent. The effectiveness of serum in mediating liposome uptake by the liver depends on liposomes size. Large liposomes appeared to be opsonized more efficiently and, therefore, taken up more by the liver than the smaller ones. The effects of liposomes size on liver uptake did not occur in the absence of serum. Treatment of serum at 56 degrees C for 30 min abolished the serum activity, suggesting the involvement of complement components. Inhibition of the hemolytic activity of complement through the alternative pathway by PS-containing liposomes suggests that components in this pathway are responsible for liposome opsonization. Liposomes containing phosphatidic acid, phosphatidylglycerol, and dicetyl phosphate compete in different degrees for serum components which mediate the liver uptake of PS-containing liposomes. These results suggest that the opsonization of liposomes by serum opsonins are the determining factors for the recognition and clearance of liposomes by the RES. Complement components are most likely involved in this process. The results presented here are relevant to the use of liposomes as drug delivery vehicle in vivo and to the PS-mediated clearance of red blood cells from the blood circulation.

Enhanced gene expression in mouse lung by prolonging the retention time of intravenously injected plasmid DNA

The effect of retention time of plasmid DNA in mouse lung on the level of transgene expression after intravenous administration was examined. Using CMV driven expression system with luciferase gene as a reporter and preinjection of free cationic liposomes into the animal as means of manipulating the retention time of plasmid DNA, we demonstrated that naked plasmid DNA is effective in transfecting cells in the lung by intravenous administration. An increase in DNA retention time in the lung results in a higher level of gene expression. Liposomes composed of cationic lipids with alkyl chains exhibited better activity than cholesterol-based cationic liposomes to retain the plasmid DNA in the lung. The level and patterns of gene expression obtained appeared similar to those seen in animals transfected by DNA–liposome complexes. These results suggest that prolonging the exposure time of DNA to the target cells in vivo may be an important strategy in achieving a high level of gene expression. Our data also introduce a possibility that the function of cationic liposomes in lipoplex-mediated transfection in vivo is to extend the interaction time of DNA with the cells.

Role of Galectin-3 in Breast Cancer Metastasis: Involvement of Nitric Oxide

We investigated the role of galectin-3 in metastasis of human breast carcinoma BT549 cells using the experimental liver metastasis model. Underlying mechanisms were then elucidated using the liver/tumor co-culture and cell culture systems. After intrasplenic injection, galectin-3 cDNA transfected BT549 cells (BT549(gal-3 wt)) formed metastatic colonies in the liver, while galectin-3 null BT549 cells (BT549(par)) did not, demonstrating that galectin-3 enhances metastatic potential. More than 90% of BT549(gal-3 wt) cells survived after 24 hours-co-culture with the liver fragments isolated following ischemia treatment. In contrast, more than half of BT549(par) cells showed metabolic death following co-culture with the liver fragments. When the liver from inducible nitric oxide synthase (iNOS) knockout mice was used, no cytotoxicity to BT549(par) cells was observed. Thus, iNOS exerts cytotoxicity on BT549(par) cells and galectin-3 can protect against iNOS-induced cytotoxicity. BT549(gal-3 wt) also exhibited enhanced survival against peroxynitrite (up to 400 micromol/L) in vitro. A single mutation in the NWGR motif of galectin-3 obliterated both metastatic capability and cell survival, indicating that the antiapoptotic function of galectin-3 is involved in enhanced metastasis. In conclusion, galectin-3 enhances the metastatic potential of BT549 cells through resistance to the products of iNOS, possibly through its bcl-2-like antiapoptotic function.

Sodium nitroprusside enhances TRAIL-induced apoptosis via a mitochondria-dependent pathway in human colorectal carcinoma CX-1 cells.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo-2L) is a recently characterized member of the family of programmed cell death-inducing ligands that includes TNF-α and CD95L (FasL). It is well known that TRAIL binds to the death signaling receptors, DR4 and DR5, and initiates the TRAIL death pathway. Activation of this pathway, mediated through a caspase cascade, causes apoptosis. In this study, we hypothesized that oxidative stress facilitates TRAIL-induced apoptosis by promoting caspase activity through cytochrome c release from mitochondria. Human colorectal carcinoma CX-1 cells were treated with various concentrations of TRAIL (12.5–200 ng/ml) and/or sodium nitroprusside (SNP; 0.03–1 mM) for 12 h. SNP, a nitric oxide donor, which had little toxic effect by itself, enhanced TRAIL-induced cytotoxicity. For example, TRAIL-induced apoptosis (200 ng/ml) was increased by a factor of 2.5-fold in the presence of 1 mM SNP. The combined treatment also caused an increase in cytochrome c release, caspase-3 activity, and PARP cleavage. Overexpression of Bcl-2 completely blocked the SNP-promoting effects, but only moderately inhibited TRAIL-induced apoptosis. Similar results were observed in the presence of hydrogen peroxide or peroxynitrite. Taken together, the present studies suggest that SNP enhances TRAIL-induced cytotoxicity by facilitating the mitochondria-mediated caspase signal transduction pathway.

Structural basis of DOTMA for its high intravenous transfection activity in mouse

Eleven structural analogues of two known cationic lipids, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP) were synthesized and utilized to evaluate the structural characteristics of DOTMA for its high intravenous transfection activity. Using a CMV-driven expression system and luciferase gene as a reporter, the transfection activity of these analogues was evaluated in mice using tail vein injection. Results concerning the structure–activity relationship with regard to the influence of the backbone, relative position between head group and the hydrophobic chains on the backbone, linkage bonds, as well as the composition of the aliphatic chains revealed that cationic lipids which give a higher in vivo transfection activity share the following structural characteristics: (1) cationic head group and its neighboring aliphatic chain being in a 1,2-relationship on the backbone; (2) ether bond for bridging the aliphatic chains to the backbone; and (3) paired oleyl chains as the hydrophobic anchor. Cationic lipids without these structural features had lower in vivo transfection activity. These structural characteristics, however, did not significantly influence their in vitro transfection activity. The contribution that cationic lipids make to the overall in vivo transfection activity is likely to be determined by the structure of DNA/lipid complexes and by the outcome of the interaction between the DNA/lipid complexes and blood components upon intravenous administration.

Liposome clearance from blood: different animal species have different mechanisms

The kinetics of blood clearance and the mechanisms of liposome uptake by the reticuloendothelial system (RES) were compared in two animal species (mice and rats). By employing an in situ liver perfusion technique with selected liposome compositions (PC/Chol, PC/Cho/PS, PC/Chol/GM1 and PC/Chol/PEG5000-PE), we demonstrated that liposomes with same lipid composition exhibited different blood circulation half-lives in different animal species. Although liver is the major organ responsible for the clearance of liposomes from blood in both animal species, the specific mechanisms differ. In mice, liposome uptake by the liver did not involve specific serum opsonins. In contrast, liposome uptake by the rat liver was strongly dependent on serum opsonins. Further, the activity of serum opsonins for a given liposome composition differed among animal species. Human serum exhibited higher opsonin activities for PC/Chol and PC/Chol/GM1 liposomes than bovine sera, while rat serum displayed a high opsonizing activity for GM1 liposomes and none for liposomes composed of PC and Chol. The opsonin activity of human serum could be removed or decreased by treatment with EGTA/Mg2+, EDTA or cobra venom factor, suggesting that the activity is likely due to complement components. It is likely that C3 of the human complement system plays an important role in mediating the uptake of liposomes by the liver.

Reconstitution of galectin-3 alters glutathione content and potentiates TRAIL-induced cytotoxicity by dephosphorylation of Akt

We investigated the role of galectin-3 in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptotic death in human breast carcinoma BT549 cells. We observed that parental galectin-3 null BT549 cells (BT549(par)) as well as control vector transfected (BT549(neo)) cells were resistant to TRAIL, while galectin-3 cDNA-transfected BT549 cells (BT549(gal-3)) were sensitive to TRAIL. Data from flow cytometry and immunoblotting analyses reveal that reconstitution of galectin-3 promoted cell death and PARP cleavage as well as caspase (-8, -9, and -3) activation during TRAIL treatment. However, unlike TRAIL treatment, galectin-3 transfectants were resistant to UV-B-induced PARP cleavage. Data from cDNA array analysis show that galectin-3 did not significantly enhance or reduce any apoptosis-related gene expression. Moreover, although galectin-3 restored pre-mRNA splicing activity and resulted in elevation of FLIPs protein, experiments with FLIPs cDNA-transfected cells show that overexpression of FLIPs did not sensitize cells to TRAIL. Interestingly, BT549(gal-3) cells demonstrated a approximately 2-fold increase in total glutathione content as well as a approximately 5-fold increase in GSSG content in comparison to BT549(par) and BT549(neo) cells, suggesting that galectin-3 overexpression may alter intraceullular oxidation/reduction reactions affecting the metabolism of glutathione and other thiols. In addition, galectin-3 overexpression inactivated Akt by dephosphorylation. Finally, overexpression of constitutively activated Akt protected BT549(gal-3) cells from TRAIL-induced cytotoxicity. Taken together, our data suggest that galectin-3-enhanced TRAIL-induced cytotoxicity is mediated through dephosphorylation of Akt, possibly through a redox-dependent process.