- Email: [email protected]
24. New Insight into Cellular Uptake of Nucleic Acids: A Role for the Nucleic Acid-Conducting Channel
NAKED DNA: METHODS study). In addition, expression of I-SCeI resulted in cleavage and excision of hAAT and hFIX expression cassettes from plasmid backbone, as established by Southern blot analyses. This suggested that covalent linkage of bacterial DNA markedly diminished longterm transgene expression in vivo. Therefore, to increase the persistence of transgenes in vivo and, thus, optimize non viral gene therapy approaches, the excision of the transgenes from plasmid backbone and the removal of bacterial DNA should be considered.
24. New Insight into Cellular Uptake of Nucleic Acids: A Role for the Nucleic AcidConducting Channel Edgar Leal-Pinto,1 Avelino Teixeira,1 Scott C. Henderson,2 Mary E. Hawkins,3 Paul E. Klotman,1 Basil Hanss.1 1 Nephrology/Medicine, Mt. Sinai School of Medicine, New York, NY, United States; 2Molecular, Cell, and Developmental Biology, Mt. Sinai School of Medicine, New York, NY, United States; 3 Pediatric Branch, National Cancer Institute, NIH, Bethesda, MD, United States. Much of the published literature suggests that receptor-mediated endocytosis is the primary mechanism of cellular uptake of oligodeoxynucleic acids (ODN). In many of these studies ODNs are labeled with a large, hydrophobic fluorophore such as FAM (a derivative of fluorescein). We have hypothesized that the presence of a large hydrophobic molecule on the ODN would change the kinetics, if not the mechanism, of uptake. To test this hypothesis we developed the methodology to examine ODN uptake in cultured cells using a new structural analog of 2-deoxyguanosine, 3-Methyl8-(2-deoxy-β-D-ribofuranosyl) isoxanthopterin (3MI). 3MI is highly fluorescent and is very closely related in structure to guanosine. As such, it does not introduce significant structural or steric changes to an ODN. For these studies, ODN was labeled with 3MI or FAM (FAM-ODN) and comparisons of uptake kinetics in LLC-PK1 cells (a renal epithelial cell line derived from pig kidney) were made using multi-photon laser scanning microscopy. The rate of 3MI-ODN uptake is significantly (p<0.05) higher than that of FAM-ODN, suggesting that FAM labeled- and 3MI labeled-ODNs are transported by different mechanisms. The kinetics of FAM-ODN uptake suggested a single carrier model with a time constant consistent with endocytosis. 3MI-ODN uptake was best fit by a multicarrier model with two components, a fast component and a slow component. Based on these data, we hypothesized that FAM labeled ODNs gain entry to the cell exclusively via an endocytic pathway whereas 3MI-ODNs enter the cell via an endocytic pathway and by a fast component that we hypothesize represents the nucleic acid channel. To test these hypotheses, uptake experiments were performed in the presence or absence of methyl-β-cyclodextrin (MβCD; 10 mM - 30 minute pre-incubation), a blocker of both clathrin- and caveolaemediated endocytosis. FAM-ODN uptake was significantly decreased when cells were preincubated with MβCD. These data suggest that FAM-ODN is transported into the cell primarily by an endocytic process. Treatment with MβCD did not inhibit the fast component of 3MI-ODN uptake whereas the slow component of uptake was completely inhibited. These data indicate that the slow component of 3MI-ODN uptake is mediated by endocytosis. To determine if the fast component of 3MI-ODN uptake is mediated by the nucleic acid channel (NACh), experiments were conducted using the antiserum GN-2640, which blocks NACh in lipid bilayer experiments. When cells were pre-treated with GN-2640 (diluted 1:50) beginning 10 minutes prior to adding 3MI-ODN, the fast component of ODN uptake was blocked, suggesting it is mediated by NACh. The slow endocytic component, however, remained intact as indicated by a gradual increase in intracellular fluorescence. Preimmune serum was without effect. In summary, these data indicate that FAM-ODN enters the cell primarily by endocytosis, whereas S10
3MI-ODN is internalized by the combined action of NACh and an endocytic pathway. These data have led us to conclude that the nucleic acid channel is significant mechanism of ODN uptake in LLC-PK1 cells.
25. Enhancement of Ultrasound-Mediated Transfection by the Combination of Cavitation Facilitation and Plasma Membrane Modification In Vitro and In Vivo Ryohei Ogawa,1 Tetsuo Nozaki,2 Go Kagiya,3 Loreto B. Feril, Jr.,1 Hideki Fuse,2 Takashi Kondo.1 1 Department of Radiological Sciences, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama, Toyama, Japan; 2Department of Urology, Toyama Medical and Pharmaceutical University, Toyama, Toyama, Japan; 3Medical Division, The Wakasa Wan Energy Research Center, Tsuruga, Fukui, Japan. We previously reported that an echo contrast agent, Levovist, enhanced the effect of ultrasound-mediated transfection (USMT) in vitro (Ultrason Sonochem, 9: 197 (2002)). The mechanism underlying USMT is possibly explained by the interaction between the effects of inertial cavitation associated with ultrasound and a response of the plasma membrane to its effects, although the real mechanism has not been completely elucidated. In this study we focused on the plasma membrane to see if altering it could enhance USMT in vitro and in vivo. PC-3 cells from a prostate cancer and T24 cells from a bladder cancer were used as target cells. A plasmid with the luciferase gene under control of a CMV promoter was used as a reporter. To modify plasma membrane, lidocaine, a local anesthetic, or heat was applied. Levovist was used to facilitate cavitation generation. In vitro, cells in a rotating polystylene tube were sonicated in a water bath at various temperatures with 1 MHz continuous ultrasound at 3 W/cm² for 20 sec. In vivo, cells were transabdominally sonicated after cell suspension was transurethrally introduced in the bladder of a 4-week old female Wister rat with 1 MHz ultrasound at 2 W/ cm² with 30% duty cycle for 60 sec. Sonicated cells were incubated at 37°C for 24 hrs before lysing and subjecting to lucifearase assay. In the case of in vitro transfection of PC-3 cells, luciferase expression increased with increasing lidocaine concentration and also increased with rising temperature only when Levovist was present. In the presence of 10 mg/ml of Levovist, we observed 17fold higher efficiency in USMT with 1 mM lidocaine and 18-fold at 44°C in comparison with that observed without lidocaine at 37°C. The increase in reporter gene expression was not due to induced promoter activity since the increase was not observed when lidocaine or heat was applied immediately after sonication. Both treatments were shown to increase plasma membrane fluidity though at different degrees. In addition, heat significantly facilitated cavitational effect determined by free radical detection on ESR, but lidocaine did not, suggesting different mechanisms of USMT enhancement by lidocaine and heat. In the case of in vivo transfection of PC-3 cells, we observed similar results to those of the in vitro study. Detected luciferase activities increased dose-dependently with Levovist (50 to 200 mg/ ml). When 1 mM lidocaine was added to the cell suspension in addition to 100 mg/ml Levovist, detected luciferase activity increased about 5-fold of that without lidocaine. Likewise, when a rat was incubated in a water bath at 42°C prior to sonication with the bladder instilled with cell suspension containing 100 mg/ml Levovist, the luciferase activity was raised to 9-fold of that sonicated without preincubation. T24 cells showed similar results though to a lesser degree.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
24. New Insight into Cellular Uptake of Nucleic Acids: A Role for the Nucleic AcidConducting Channel Edgar Leal-Pinto,1 Avelino Teixeira,1 Scott C. Henderson,2 Mary E. Hawkins,3 Paul E. Klotman,1 Basil Hanss.1 1 Nephrology/Medicine, Mt. Sinai School of Medicine, New York, NY, United States; 2Molecular, Cell, and Developmental Biology, Mt. Sinai School of Medicine, New York, NY, United States; 3 Pediatric Branch, National Cancer Institute, NIH, Bethesda, MD, United States. Much of the published literature suggests that receptor-mediated endocytosis is the primary mechanism of cellular uptake of oligodeoxynucleic acids (ODN). In many of these studies ODNs are labeled with a large, hydrophobic fluorophore such as FAM (a derivative of fluorescein). We have hypothesized that the presence of a large hydrophobic molecule on the ODN would change the kinetics, if not the mechanism, of uptake. To test this hypothesis we developed the methodology to examine ODN uptake in cultured cells using a new structural analog of 2-deoxyguanosine, 3-Methyl8-(2-deoxy-β-D-ribofuranosyl) isoxanthopterin (3MI). 3MI is highly fluorescent and is very closely related in structure to guanosine. As such, it does not introduce significant structural or steric changes to an ODN. For these studies, ODN was labeled with 3MI or FAM (FAM-ODN) and comparisons of uptake kinetics in LLC-PK1 cells (a renal epithelial cell line derived from pig kidney) were made using multi-photon laser scanning microscopy. The rate of 3MI-ODN uptake is significantly (p<0.05) higher than that of FAM-ODN, suggesting that FAM labeled- and 3MI labeled-ODNs are transported by different mechanisms. The kinetics of FAM-ODN uptake suggested a single carrier model with a time constant consistent with endocytosis. 3MI-ODN uptake was best fit by a multicarrier model with two components, a fast component and a slow component. Based on these data, we hypothesized that FAM labeled ODNs gain entry to the cell exclusively via an endocytic pathway whereas 3MI-ODNs enter the cell via an endocytic pathway and by a fast component that we hypothesize represents the nucleic acid channel. To test these hypotheses, uptake experiments were performed in the presence or absence of methyl-β-cyclodextrin (MβCD; 10 mM - 30 minute pre-incubation), a blocker of both clathrin- and caveolaemediated endocytosis. FAM-ODN uptake was significantly decreased when cells were preincubated with MβCD. These data suggest that FAM-ODN is transported into the cell primarily by an endocytic process. Treatment with MβCD did not inhibit the fast component of 3MI-ODN uptake whereas the slow component of uptake was completely inhibited. These data indicate that the slow component of 3MI-ODN uptake is mediated by endocytosis. To determine if the fast component of 3MI-ODN uptake is mediated by the nucleic acid channel (NACh), experiments were conducted using the antiserum GN-2640, which blocks NACh in lipid bilayer experiments. When cells were pre-treated with GN-2640 (diluted 1:50) beginning 10 minutes prior to adding 3MI-ODN, the fast component of ODN uptake was blocked, suggesting it is mediated by NACh. The slow endocytic component, however, remained intact as indicated by a gradual increase in intracellular fluorescence. Preimmune serum was without effect. In summary, these data indicate that FAM-ODN enters the cell primarily by endocytosis, whereas S10
3MI-ODN is internalized by the combined action of NACh and an endocytic pathway. These data have led us to conclude that the nucleic acid channel is significant mechanism of ODN uptake in LLC-PK1 cells.
25. Enhancement of Ultrasound-Mediated Transfection by the Combination of Cavitation Facilitation and Plasma Membrane Modification In Vitro and In Vivo Ryohei Ogawa,1 Tetsuo Nozaki,2 Go Kagiya,3 Loreto B. Feril, Jr.,1 Hideki Fuse,2 Takashi Kondo.1 1 Department of Radiological Sciences, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama, Toyama, Japan; 2Department of Urology, Toyama Medical and Pharmaceutical University, Toyama, Toyama, Japan; 3Medical Division, The Wakasa Wan Energy Research Center, Tsuruga, Fukui, Japan. We previously reported that an echo contrast agent, Levovist, enhanced the effect of ultrasound-mediated transfection (USMT) in vitro (Ultrason Sonochem, 9: 197 (2002)). The mechanism underlying USMT is possibly explained by the interaction between the effects of inertial cavitation associated with ultrasound and a response of the plasma membrane to its effects, although the real mechanism has not been completely elucidated. In this study we focused on the plasma membrane to see if altering it could enhance USMT in vitro and in vivo. PC-3 cells from a prostate cancer and T24 cells from a bladder cancer were used as target cells. A plasmid with the luciferase gene under control of a CMV promoter was used as a reporter. To modify plasma membrane, lidocaine, a local anesthetic, or heat was applied. Levovist was used to facilitate cavitation generation. In vitro, cells in a rotating polystylene tube were sonicated in a water bath at various temperatures with 1 MHz continuous ultrasound at 3 W/cm² for 20 sec. In vivo, cells were transabdominally sonicated after cell suspension was transurethrally introduced in the bladder of a 4-week old female Wister rat with 1 MHz ultrasound at 2 W/ cm² with 30% duty cycle for 60 sec. Sonicated cells were incubated at 37°C for 24 hrs before lysing and subjecting to lucifearase assay. In the case of in vitro transfection of PC-3 cells, luciferase expression increased with increasing lidocaine concentration and also increased with rising temperature only when Levovist was present. In the presence of 10 mg/ml of Levovist, we observed 17fold higher efficiency in USMT with 1 mM lidocaine and 18-fold at 44°C in comparison with that observed without lidocaine at 37°C. The increase in reporter gene expression was not due to induced promoter activity since the increase was not observed when lidocaine or heat was applied immediately after sonication. Both treatments were shown to increase plasma membrane fluidity though at different degrees. In addition, heat significantly facilitated cavitational effect determined by free radical detection on ESR, but lidocaine did not, suggesting different mechanisms of USMT enhancement by lidocaine and heat. In the case of in vivo transfection of PC-3 cells, we observed similar results to those of the in vitro study. Detected luciferase activities increased dose-dependently with Levovist (50 to 200 mg/ ml). When 1 mM lidocaine was added to the cell suspension in addition to 100 mg/ml Levovist, detected luciferase activity increased about 5-fold of that without lidocaine. Likewise, when a rat was incubated in a water bath at 42°C prior to sonication with the bladder instilled with cell suspension containing 100 mg/ml Levovist, the luciferase activity was raised to 9-fold of that sonicated without preincubation. T24 cells showed similar results though to a lesser degree.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy