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Microtubule Involvement in the Intracellular Dynamics for Gene Transfection Mediated by Cationic Liposomes

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Microtubule Involvement in the Intracellular Dynamics for Gene Transfection Mediated by Cationic Liposomes Gene Therapy (2001) 8, 1669–1673 2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00 www.nature.com/gt BRIEF COMMUNICATION Microtubule involvement in the intracellular dynamics for gene transfection mediated by cationic liposomes S Hasegawa, N Hirashima and M Nakanishi Graduate School of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan The effects of microtubule polymerization on liposome- tered throughout the cytoplasm and they did not colocalize. mediated gene transfection were investigated by confocal In the presence of taxol, microtubules were stabilized and laser scanning microscopy in target living cells. Both nocod- several focal regions, like the MTOC, were formed. Lyso- azole and taxol apparently increased the efficiency of gene somes resided around the nucleus, while liposomes were transfection. Lipofection with fluorescence-labeled cationic trapped in microtubules. Under these conditions, neither liposomes in a COS-7 cell expressing yellow fluorescent pro- liposomes nor DNA colocalized with lysosomes. These tein (YFP)-tagged tubulin revealed that the liposomes were results demonstrated that the liposome–DNA complexes are transported along microtubules to lysosomes which are col- transported to lysosomes by a microtubule-mediated path- ocalized with the microtubule organizing center (MTOC). way, and the effects of nocodazole and taxol on transfection Nocodazole disrupted microtubules and produced a uniform efficiency can be explained by failure of the transport of the distribution of YFP-tagged tubulin in the cytoplasm. Under liposome–DNA complexes to lysosomes where DNAs are these conditions, both liposomes and lysosomes were scat- degraded. Gene Therapy (2001) 8, 1669–1673. Keywords: cationic liposome; cationic cholesterol; microtubule; gene transfection; laser scanning confocal microscopy Among various methods for gene transfection, lipofec- some–DNA complexes and microtubules in living cul- tion using cationic liposomes is considered to be a prom- tured cells, and showed that microtubules are involved ising way to deliver foreign gene to the target cells. Quite in the intracellular transport of the exogenous liposome– a few cationic liposomes for lipofection have been DNA complex. developed,1–3 since efficient vectors such as DOTMA In order to investigate the contribution of microtubules (Lipofectin)4 and DC-Chol5 have been reported. To to the transport of exogenous DNA and liposomes, we increase efficiency, we recently introduced novel cationic tested the effects of nocodazole and taxol on transfection liposomes into this field. The new cationic liposomes efficiency in COS-7 cells by luciferase assay. For transfec- with a cationic cholesterol derivative containing hydroxy tion with luciferase plasmids using cationic lipids, pGL3 amino head group, 3β[N-(2-hydroxyethylaminoethane)- (Promega, Madison, WI, USA) were complexed with carbamoyl]-cholestene (HyC-Chol), has greater efficiency liposomes consisting of DOPE (dioleoylphos- than DOTMA and DC-Chol.6,7 To improve the efficiency phatidylethanolamine; Avanti Polar Lipids, Alabaster, of the transfection, it is essential to elucidate the mech- AL, USA) and HyC-Chol (DOPE:HyC-Chol = 2:3 anism not only as to how exogenous DNA is taken up (mol/mol)) to form liposome–DNA complexes. Lipo- and transported to the nucleus, but also how DNA some-DNA complexes were incubated with the cells for degrades, since inhibition of the degradation of exogen- 4 h at 37°C, and then the cells were washed and cultured ous DNA could contribute to the enhancement of trans- in DMEM for another 24 h at 37°C, followed by a lucifer- fection efficiency, as well as facilitation of the nuclear ase assay. When cells were treated with nocodazole, translocation. Liposome–DNA complex is taken up by which disrupts microtubules, the efficiency of transfec- endocytosis, and the internalized DNA is released from tion increased dose-dependently and about doubled at 10 endosomes by their acidification.8–10 However, the details ␮m, as shown in Figure 1a. Taxol, which stabilizes of intracellular events during lipofection are still microtubules, also enhanced transfection efficiency in a unknown. Although many lines of evidence suggest that dose-dependent manner (Figure 1b). The concentrations microtubules are responsible for the intracellular traf- at which these reagents had effects were within the range ficking of materials internalized by endocytosis,11,12 no of their working concentrations to affect microtubules direct observation as to how microtubules are involved in specifically. Therefore, the results suggest that modifi- the intracellular dynamics of exogenous liposome–DNA cation of the polymerization states of microtubules complex has been reported. In the present study, there- changes the efficiency of gene transfection. To examine fore, we undertook simultaneous visualization of lipo- the possibility that these reagents increased the ability of cells to interact with exogenous liposome–DNA com- plexes, cells were incubated with the liposome–DNA Correspondence: M Nakanishi complexes containing NBD-PE (N-(7-nitrobenz-2-oxa-1,3- Received 10 May 2001; accepted 24 August 2001 diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phospho- Microtubule involvement for gene transfection S Hasegawa et al 1670 Figure 1 Effects of nocodazole and taxol on transfection efficiency and uptake of liposome-DNA complexes. Cells were preincubated with nocodazole or taxol for 60 min before incubation with liposome–DNA complexes. (a, b) The luciferase assay was carried out using a picagene luciferase assay kit (Toyo Ink, Tokyo, Japan). The intensity of chemiluminescence was measured with a luminometer (TD-20/20; Turner Designs, Sunnyvale, CA, USA) and values were normalized to the amount of protein. Nocodazole (a) and taxol (b) increased the transfection efficiency in a dose-dependent manner. (c) Cells pretreated with nocodazole or taxol were incubated with DNA-liposome complexes that contained NBD-labeled PE for 4 h. After washing, cells bearing NBD fluorescence were detected by a flow cytofluorometer. Data from cells treated with nocodazole (green) and taxol (blue) are almost superimposable on that of control (red). Yellow line shows background fluorescence (autofluorescence) obtained from cells without NBD-labeled PE. ethanolamine; Molecular Probes, Eugene, OR, USA), and cells bearing NBD fluorescence were detected by flow cytometry at 4 h. As shown in Figure 1c, neither nocoda- zole nor taxol changed the amount of liposome–DNA complexes loaded in COS-7 cells. This is consistent with a previous report concluding that actin, not the microtu- bules, is responsible for the initial binding and uptake by endocytosis,13 and suggests that these two reagents enhanced the transfection efficiency by modifying the intracellular trafficking of exogenous DNA and lipo- somes. Thus, we investigated the intracellular dynamics of cationic liposome–DNA complex and the distribution of microtubules in COS-7 cells. To visualize the distribution of liposomes and DNA Figure 2 Distribution of DNA and liposomes after transfection with introduced as liposome–DNA complexes, DNA and lipo- DNA-liposome complexes. Cells were incubated with complex of FITC- somes were labeled with FITC and rhodamine, respect- labeled c-myc antisense oligonucleotides and rhodamine-PE labeled lipo- ively. Figure 2 shows the distribution DNA and lipo- somes. At 4 h after incubation, difference interference contrast (DIC; left) somes at 4 h after incubation of COS-7 cells with and fluorescent (right) images were obtained with a confocal laser scan- liposome–DNA complexes. Liposomes and DNA were ning microscope (LSM-510, Zeiss). DNA and liposomes appeared green colocalized in the cytoplasm and extracellular medium, and red, respectively. White lines in fluorescent images represent the out- line of the cell. Liposomes and DNA were colocalized in the cytoplasm while only DNA was found in the nucleus. This suggests and extracellular medium (in yellow), while only DNA was found in the that internalized liposome and DNA are transported as nucleus (in green). a complex in the cytoplasm, and DNA is released from liposomes to enter the nucleus. To investigate the spatial Figure 3 Double staining of microtubules and liposomes in a living cell. Rhodamine-PE labeled liposomes complexed with DNA were introduced into cells, which stably expressed YFP-tubulin. Confocal images of liposomes and microtubules are shown at the indicated number of hours of incubation with liposome-DNA complexes. YFP-tubulin and rhodamine-PE labeled liposomes are green and red, respectively. Most liposomes moved along the microtubules (yellow) and converged on the microtubule organizing center (MTOC) within 4 h of incubation. Gene Therapy Microtubule involvement for gene transfection S Hasegawa et al 1671 Figure 4 Effects of nocodazole and taxol on the distribution of tubulin, liposomes and lysosomes. (a) Tubulin and liposomes were stained as in Figure 3. Cells were pre-incubated with nocodazole (upper images) or taxol (lower images). In cells treated with nocodazole, the filamentous structures of microtubules disappeared and liposomes were scattered throughout the cytoplasm even at 4 h after transfection. However, in taxol-treated cells, excess polymerization of tubulin gave rise to the formation of several microtubule-rich regions where liposomes were trapped. (b) Lysosomes were stained by LysoTracker Red in a cell expressing YFP-tubulin. Lysosomes (red) were colocalized
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