Cationic lipids involved in gene transfer mobilize intracellular calcium

Figures & data

Figure 1.  (A) Effect of different types of cationic liposomes on [Ca^2 +]_i and comparison of [Ca^2 +]_i rise induced by diC14-amidine and ADP in K562 cells. Cells (1×10⁶ cells/ml) resuspended in D-PBS medium at 1 mg/ml glucose in the presence of 1 mM Ca^2 + were loaded with Fluo-3-AM for 30 min at 37°C, and then exposed (arrow) to the indicated concentrations of cationic liposome (37 µM). The insert graph shows [Ca^2 +]_i changes induced by diC14-amidine (37 µM) and ADP (20 µM). Intracellular Ca^2 + concentration changes were measured at 37°C using fluorescence spectroscopy as described in Materials and Methods. The same experiment was repeated three times and showed identical results. (B) Dose-dependent intracellular Ca^2 + increase induced by diC14-amidine in K562 cells. Cells (1×10⁶ cells/ml) resuspended in D-PBS medium at 1 mg/ml glucose in the presence of 1 mM Ca^2 + were loaded with Fluo-3-AM for 30 min at 37°C and then exposed (arrow) to the indicated concentrations of diC14-amidine. The insert graph shows [Ca^2 +]_i changes induced by diC14-amidine (37 µM). Intracellular Ca^2 + concentration changes were measured at 37°C using fluorescence spectroscopy as described in Materials and Methods. The same experiment was repeated three times and showed identical results.

Figure 2.  Effect of cationic lipid/DNA complexes charged positively on [Ca^2 +]_i increase in K562 cells. Cells (1×10⁶ cells/ml) resuspended in D-PBS medium at 1 mg/ml glucose in the presence of 1 mM Ca^2 + were loaded with Fluo-3-AM for 30 min at 37°C, and then exposed (arrow) to diC14-amidine/DNA (weight ratio) (Figure 2A) or DOTAP/DNA (weight ratio) (Figure 2B). Complexes were formed as indicated in Materials and Methods. Final concentration of DNA was 5 µg/ml. Intracellular Ca^2 + concentration changes were measured at 37°C using fluorescence spectroscopy as described in Materials and Methods. The same experiments were repeated three times and showed identical results.

Figure 3.  Contribution of intra and extracellular Ca^2 + to [Ca^2 +]_i induced by diC14-amidine/DNA complexes and diC14-amidine liposomes in K562 cells. Cells (1×10⁶ cells/ml) resuspended in D-PBS medium at 1 mg/ml glucose in the presence of 1 mM Ca^2 + were loaded with Fluo-3-AM for 30 min at 37°C. After loading, cells were rinsed with D-PBS without Ca^2 + and suspended in 1 ml of the same medium. At time = 100 s, calcium (1 mM final) or EGTA (20 µM final) was added. At time = 200 s, diC14-amidine/DNA complex at a weight ratio 2:1 (5 µg DNA) (Figure 3A) or free diC14-amidine liposomes (37 µM) (Figure 3B) was added and [Ca^2 +]_i changes were measured at 37°C using fluorescence spectroscopy as described in Materials and Methods. The same experiments were repeated three times and showed identical results.

Figure 4.  Effect of diC14-amidine on [Ca^2 +]_i in TG treated K562 cells. Cells (1×10⁶ cells/ml) resuspended in D-PBS medium at 1 mg/ml glucose in the presence of 1 mM Ca^2 + were loaded with Fluo-3-AM for 30 min at 37°C, and then incubated in Ca^2 +-free medium (no Ca^2 + plus 20 µM EGTA). At time = 200 s, 5 µl of 65 µg/ml TG in DMSO (grey curve) or the same volume of DMSO (control, black curve) was added. At t = 500 s, diC14-amidine (37 µM) was added. Intracellular Ca^2 + concentration changes were measured at 37°C using fluorescence spectroscopy as described in Materials and Methods. The same experiment was repeated three times and showed identical results.

Figure 5.  (A) Effect of U73122 on the [Ca^2 +]_i increase induced by diC14-amidine/DNA complexes and diC14-amidine liposomes in K562 cells. Cells (1×10⁶ cells/ml) resuspended in D-PBS medium at 1 mg/ml glucose in the presence of 1 mM Ca^2 + were loaded with Fluo-3-AM for 30 min at 37°C, and pretreated with the PLC inhibitor U73122 (10 µM) for 10 min before the addition of diC14-amidine/DNA complex at a 2:1 weight ratio (5 µg DNA) or 37 µM diC14-amidine liposome as indicated by the arrow. (B) Effect of U73122 on the [Ca^2 +]_i increase induced by DOTAP/DNA complexes and DOTAP liposomes in K562 cells. Cells (1×10⁶ cells/ml) resuspended in D-PBS medium at 1 mg/ml glucose in the presence of 1 mM Ca^2 + were loaded with Fluo-3-AM for 30 min at 37°C, and pretreated with the phospholipase C inhibitor U73122 (10 µM) for 10 min before addition of DOTAP/DNA complex at a 2:1 weight ratio (5 µg DNA) or 37 µM DOTAP liposome as indicated by the arrow. (C) Comparison of the effect of U73122 and its isomer U73343 inactive on PLC. K562 cells (1×10⁶ cells/ml) resuspended in D-PBS medium at 1 mg/ml glucose in the presence of 1 mM Ca^2 + were loaded with Fluo-3-AM for 30 min at 37°C, and pretreated with U73343 (10 µM) or U73122 (10 µM) for 10 min before addition of 37 µM diC14-amidine liposome as indicated by the arrow. Intracellular Ca^2 + concentration changes were measured at 37°C using fluorescence spectroscopy as described in Materials and Methods. The same experiments were repeated three times and showed identical results.

Figure 6.  (A) Comparison of [Ca^2 +]_i increase induced by neutral and negatively charged liposomes in K562 cells. Cells (1×10⁶ cells/ml) resuspended in D-PBS medium at 1 mg/ml glucose in the presence of 1 mM Ca^2 + were loaded with Fluo-3-AM for 30 min at 37°C. DiC14-amidine (positive), DMPC (neutral) and DMPC/DMPG 1:3 (negative) liposomes were added (arrow) at a 37 µM concentration. (B) and (C) Effect of diC14-amidine, lipofectamine and the corresponding headgroups N-t-butyl-N′-isopropyl-3-isopropylaminopropionamidine (diisopropylamidine) and spermine respectively on [Ca^2 +]_i increase in K562 cells. Cells (1×10⁶ cells/ml) resuspended in D-PBS medium at 1 mg/ml glucose in the presence of 1 mM Ca^2 + were loaded with Fluo-3-AM for 30 min at 37°C. DiC14-amidine, lipofectamine, diisopropylamidine, and spermine were added (arrow) at a 37 µM concentration. Intracellular Ca^2 + concentration changes were measured at 37°C using fluorescence spectroscopy as described in Materials and Methods. The same experiments were repeated three times and showed identical results.