Efficient Transfer of Synthetic Ribozymes into Cells Using Hemagglutinating Virus of Japan (HVJ)-Cationic Liposomes
1997; Elsevier BV; Volume: 272; Issue: 43 Linguagem: Inglês
10.1074/jbc.272.43.27099
ISSN1083-351X
AutoresIsao Kitajima, Naohiro Hanyu, Yasuko Soejima, Ryuki Hirano, Satoko Arahira, Shoji Yamaoka, Ryo Yamada, Ikuro Maruyama, Yasufumi Kaneda,
Tópico(s)Vector-Borne Animal Diseases
ResumoWe investigated the usefulness of ribozymes in inhibiting the expression of human T-cell leukemia virus type I (HTLV-I) gene. Two hammerhead ribozymes that were against HTLV-Irex (RR) and tax (TR) mRNA were synthesized. Both ribozymes were sequence-specific in the in vitro cleavage analysis of run-off transcripts fromtax/rex cDNA. Intracellular activities of the ribozymes were studied in HTLV-I tax cDNA-transfected rat embryonic fibroblasts (Rat/Tax cells), which expressed the Tax but not Rex. Ribozymes were delivered into cells using anionic or cationic liposomes fused with hemagglutinating virus of Japan (HVJ). Cellular uptake of ribozymes complexed with HVJ-cationic liposomes was 15–20 times higher cellular uptake than naked ribozymes, and 4–5 times higher than that of ribozymes complexed with HVJ-anionic liposomes. HVJ-cationic liposomes promoted accumulation of ribozymes in cytoplasm and accelerated transport to the nucleus. Tax protein levels were decreased about 957 and were five times lower when the same amount of TR was introduced into the cells using HVJ-cationic, rather than HVJ-anionic liposomes. Inactive ribozyme and tax antisense oligodeoxynucleotides reduced Tax expression by about 207, whereas RR and tax sense oligodeoxynucleotides had no effect. These results suggest that the ribozymes' effect against taxmRNA was sequence-specific, and HVJ-cationic liposomes can be useful for intracellular introduction of ribozymes. We investigated the usefulness of ribozymes in inhibiting the expression of human T-cell leukemia virus type I (HTLV-I) gene. Two hammerhead ribozymes that were against HTLV-Irex (RR) and tax (TR) mRNA were synthesized. Both ribozymes were sequence-specific in the in vitro cleavage analysis of run-off transcripts fromtax/rex cDNA. Intracellular activities of the ribozymes were studied in HTLV-I tax cDNA-transfected rat embryonic fibroblasts (Rat/Tax cells), which expressed the Tax but not Rex. Ribozymes were delivered into cells using anionic or cationic liposomes fused with hemagglutinating virus of Japan (HVJ). Cellular uptake of ribozymes complexed with HVJ-cationic liposomes was 15–20 times higher cellular uptake than naked ribozymes, and 4–5 times higher than that of ribozymes complexed with HVJ-anionic liposomes. HVJ-cationic liposomes promoted accumulation of ribozymes in cytoplasm and accelerated transport to the nucleus. Tax protein levels were decreased about 957 and were five times lower when the same amount of TR was introduced into the cells using HVJ-cationic, rather than HVJ-anionic liposomes. Inactive ribozyme and tax antisense oligodeoxynucleotides reduced Tax expression by about 207, whereas RR and tax sense oligodeoxynucleotides had no effect. These results suggest that the ribozymes' effect against taxmRNA was sequence-specific, and HVJ-cationic liposomes can be useful for intracellular introduction of ribozymes. The discovery of RNA molecules with sequence-specific RNA-cleaving properties, called ribozymes, led to investigations of their potential use as specific inhibitors of gene expression (1Kruger K. Grabowski P.J. Zaung A.J. Sands J. Gottschling D.E. Cech T.R. Cell. 1982; 31: 147-157Abstract Full Text PDF PubMed Scopus (1569) Google Scholar, 2Guerrier-Takada C. Gardiner K. Marsh T. Pace N. Altman S. Cell. 1983; 35: 849-857Abstract Full Text PDF PubMed Scopus (2037) Google Scholar, 3Cech T. Bass B. Annu. Rev. Biochem. 1986; 55: 599-629Crossref PubMed Google Scholar). Several ribozyme configurations have been identified, of which the 舠hammerhead舡 (4Forster A.C. Symons R.H. Cell. 1987; 49: 211-220Abstract Full Text PDF PubMed Scopus (564) Google Scholar) and 舠hairpin舡 (5Hampel A. Tritz R. Hicks M. Cruz P. Nucleic Acids Res. 1990; 18: 299-304Crossref PubMed Scopus (212) Google Scholar) structures are the simplest, and most suitable, for biomedical applications (6Sarver N. Antisense Res. Dev. 1991; 1: 373-378Crossref PubMed Scopus (7) Google Scholar, 7Kijima H. Ishida H. Ohkawa T. Kashani-Sabet M. Scanlon K.J. Pharmacol. Ther. 1995; 68: 247-264Crossref PubMed Scopus (55) Google Scholar, 8Uhlenbeck O.C. Nature. 1987; 328: 596-600Crossref PubMed Scopus (869) Google Scholar). Several factors appear to contribute to the intracellular efficacy of ribozymes and thus the success of ribozyme gene therapy. Most importantly, the ribozyme must co-localize with its molecular target in the appropriate cellular compartment and must be present in a sufficiently high concentration to promote hybridization. Previous studies have generally assessed the catalytic activity of ribozymes in cell-free assay systems (6Sarver N. Antisense Res. Dev. 1991; 1: 373-378Crossref PubMed Scopus (7) Google Scholar, 7Kijima H. Ishida H. Ohkawa T. Kashani-Sabet M. Scanlon K.J. Pharmacol. Ther. 1995; 68: 247-264Crossref PubMed Scopus (55) Google Scholar, 8Uhlenbeck O.C. Nature. 1987; 328: 596-600Crossref PubMed Scopus (869) Google Scholar). The present study describes an experimental system that allows one to assess the effects of ribozymes in living cells. The in vivo application of ribozymes will depend on the availability of efficient delivery methods. The methods of gene transfer are classified as viral or non-viral. Ribozymes generally have been introduced into cells via a viral infection or the transfection of expression vectors (6Sarver N. Antisense Res. Dev. 1991; 1: 373-378Crossref PubMed Scopus (7) Google Scholar, 9Xing Z. Whitton J.L. J. Virol. 1992; 66: 1361-1369Crossref PubMed Google Scholar, 10Wong-Staal F. Adv. Drug Deliver. Rev. 1995; 17: 363-368Crossref Scopus (8) Google Scholar). Viral vectors can potentially lead to the incorporation of ribozyme-encoding genes into the cellular chromosomes, thereby allowing their permanent expression, and many studies now are aimed at developing suitable vectors that present a minimal associated risk to the host. Alternatively, ribozymes can be delivered to the cells by non-viral methods, such as lipophilic vesicles (liposomes) and cholesterol (6Sarver N. Antisense Res. Dev. 1991; 1: 373-378Crossref PubMed Scopus (7) Google Scholar). We recently developed a highly efficient method for gene transfer that involves the entrapment of DNA or RNA using hemagglutinating virus of Japan (HVJ, 1The abbreviations used are: HVJ, hemagglutinating virus of Japan; FITC, fluorescein isothiocyanate; HTLV-I, human T-cell leukemia virus type I; mAb, monoclonal antibody; ODN, oligodeoxynucleotide; PBS, phosphate-buffered saline; PI, propidium iodide; Rat/Tax cell, tax-expressing rat embryonic fibroblast; RR, ribozyme against HTLV-I rex mRNA; RC, inactive control ribozyme derived from RR; TR, ribozyme against HTLV-Itax mRNA; TC, inactive control ribozyme derived from TR; TRITC, tetramethylrhodamine isothiocyanate; bp, base pair(s). Sendai virus) to enhance the fusion of anionic liposomes to cell membranes (11Kaneda Y. Uchida T. Kim J. Ishiura M. Okada Y. Exp. Cell Res. 1987; 173: 56-69Crossref PubMed Scopus (92) Google Scholar, 12Kaneda Y. Iwai K. Uchida T. Science. 1989; 243: 375-379Crossref PubMed Scopus (424) Google Scholar). However, especially in cultured cells, the level of transgene expression achieved with this method is somewhat lower than that obtained with some of the viral vectors. We then improved this gene delivery system using cationic lipids for the liposomes (13Saeki Y. Matsumoto N. Nakano Y. Mori M. Awai K. Kaneda Y. Hum. Gene Ther. 1997; (in press)PubMed Google Scholar). In the present study, we compared the effects of ribozymes that had been transferred into living cells, using anionic or cationic liposomes. Ribozymes may become useful molecular therapies for several diseases of humans, including human T-cell leukemia virus type I (HTLV-I) infection, which is etiologically associated with adult T-cell leukemia (14Yoshida M. Miyoshi I. Hinuma Y. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 2031-2035Crossref PubMed Scopus (1722) Google Scholar, 15Sodroski J. Rosen C. Goh W.C. Haseltine W. Science. 1985; 228: 1430-1434Crossref PubMed Scopus (232) Google Scholar). The HTLV-I Tax protein has oncogenic properties that may play a key role in tumorigenesis (16Nerenberg M. Hinrichs S.H. Reynolds R.K. Khoury G. Jay G. Science. 1987; 237: 1324-1329Crossref PubMed Scopus (420) Google Scholar, 17Tanaka A. Takahashi C. Yamaoka S. Nosaka T. Maki M. Hatanaka M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1071-1075Crossref PubMed Scopus (369) Google Scholar). We previously evaluated gene therapy for the treatment of HTLV-I-related diseases usingtax antisense oligodeoxynucleotides (ODNs) (18Kitajima I. Shinohara T. Minor T. Bibbs L. Bilakovics J. Nerenberg M. J. Biol. Chem. 1992; 267: 25881-25888Abstract Full Text PDF PubMed Google Scholar, 19Kitajima I. Shinohara T. Bilakovics J. Brown D.A. Xu X. Nerenberg M. Science. 1992; 258: 1792-1795Crossref PubMed Scopus (262) Google Scholar, 20Kitajima I. Kawahara K. Hahyu N. Shin H. Tokioka T. Soejima Y. Tstutsui J. Ozawa M. Shimayama T. Maruyama I. J. Cell Sci. 1996; 109: 609-617PubMed Google Scholar). In the present study, we describe an approach involving ribozyme-mediated cleavage of HTLV-I tax/rex mRNA. We investigated whether ribozymes can cleave their target RNAs in the cells and whether HVJ-cationic liposome-mediated gene transfer allows efficient introduction of ribozymes into living cells. Ribozymes were chemically synthesized on a 1-ॖmol scale using a DNA synthesizer (model 8909 Expedite System, PerSeptive Biosystems, Framingham, MA). We generated two hammerhead ribozymes that targeted the tax/rex mRNA based on previously published sequence information (21Seiki M. Hattori S. Hirayama Y. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3618-3622Crossref PubMed Scopus (1143) Google Scholar). The ODNs were designed to bracket thetax and rex AUGs, which has been shown to inhibit HTLV-I Tax protein expression in experiments using antisense ODNs (18Kitajima I. Shinohara T. Minor T. Bibbs L. Bilakovics J. Nerenberg M. J. Biol. Chem. 1992; 267: 25881-25888Abstract Full Text PDF PubMed Google Scholar, 19Kitajima I. Shinohara T. Bilakovics J. Brown D.A. Xu X. Nerenberg M. Science. 1992; 258: 1792-1795Crossref PubMed Scopus (262) Google Scholar, 20Kitajima I. Kawahara K. Hahyu N. Shin H. Tokioka T. Soejima Y. Tstutsui J. Ozawa M. Shimayama T. Maruyama I. J. Cell Sci. 1996; 109: 609-617PubMed Google Scholar). The ribozyme targeting the rex mRNA (RR) had the sequence 5′-GSGSGSCCUCCCUG*AUGAGGCCGAAAGGCCGAAACGGGUSCSUS-3′. It cleaves the mRNA at position 5139 of the HTLV-I genome. The ribozyme targeting tax mRNA (TR), which cleaves the mRNA at position 7308 of the HTLV-I genome, had the sequence 5′-ASCSCSCUGGGCUG*AUGAGGCCGAAAGGCCGAAAGUGGGSCSCS-3′ (Fig. 1 A). The HTLV-I-related sequences are underlined. As described previously by Ruffner et al. (22Ruffner D.E. Stormo G.D. Uhlenbeck O.C. Biochemistry. 1990; 29: 10695-10702Crossref PubMed Scopus (449) Google Scholar), we also designed inactive control ribozymes for RR (labeled RC) and TR (labeled TC), which inactivate the hammerhead motif by replacing G5with A (indicated by *). Each ribozyme contains phosphorothioate-modified nucleotides (indicated by 舠s舡). The 5′ end of each ribozyme was labeled with fluorescein isothiocyanate (FITC) using the FluoroPrime reagent (Pharmacia Biotech, Uppsala, Sweden). Total cellular RNA was isolated from the HTLV-I-infected T-cell line, MT-2 cells (gift of Prof. I. Miyoshi, Kochi Medical School) by the acid guanidium thiocyanate-phenol-chloroform extraction method (23Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63184) Google Scholar). HTLV-Itax cDNA from the MT-2 cells was obtained by polymerase chain reaction using HTLV-I tax/rex-specific primers as described previously (24Kitajima I. Yamamoto K. Sato K. Nakajima T. Maruyama I. Osame M. Nishioka K. J. Clin. Invest. 1991; 88: 1315-1322Crossref PubMed Scopus (110) Google Scholar). The plasmid pGEM-T was purchased from Promega Corp. (Madison, WI). The run-off transcription vector, pGEM-T-tax, was obtained by cloning the 220-bp HTLV-Itax cDNA (corresponding to HTLV-I bp 5095–7357, Fig.1 A) using the NcoI site in the multiple cloning region of pGEM-T. Run-off transcription was performed in the presence of 50 ng of pGEM-T-tax plasmid linearized withBamHI, pGEM-T-tax plasmid, 500 ॖmof each dNTP, 40 mm Tris-HCl, pH 8.0, 10 mmMgCl2, 5 mm dithiothreitol, digoxigenin-labeled dUTP, and T7 RNA polymerase. After incubation at 37 °C for 120 min, the reaction was terminated by adding stop solution (957 formamide and 10 mm EDTA, pH 8.0). The cleavage kinetics of the ribozymes were determined using tax mRNA obtained by run-off transcription from pGEM-T-tax. The cleavage reactions contained 2 pmol/ml tax mRNA in 50 mm Tris-HCl, pH 8.0, 0–25 mmMgCl2, and 0.2–20 pmol/ॖl ribozyme. After incubation for 1 h at 37 °C, the reactions were stopped by adding 100 mm EDTA. The samples were heated for 5 min at 65 °C, placed immediately on ice, loaded onto a 107 polyacrylamide, 8m urea gel, and transferred to an Immobilon-S membrane (Millipore Corp., Bedford, MA). The cleavage products were detected using an anti-digoxigenin antibody conjugated with alkaline phosphatase and visualized using an enzyme-linked color reaction (Boehringer Mannheim Corp., Mannheim, Germany). HVJ-cationic liposomes were prepared as described elsewhere (12Kaneda Y. Iwai K. Uchida T. Science. 1989; 243: 375-379Crossref PubMed Scopus (424) Google Scholar, 13Saeki Y. Matsumoto N. Nakano Y. Mori M. Awai K. Kaneda Y. Hum. Gene Ther. 1997; (in press)PubMed Google Scholar, 25Gao X. Huang L. Biochem. Biophys. Res. Commun. 1991; 179: 280-285Crossref PubMed Scopus (904) Google Scholar). In brief, a lipid mixture that contained 6 mg of phosphatidylcholine, 3 mg of cholesterol, and 0.75 mg of 3ॆ-[N-(N′,N-dimethylaminoethane)carbamoyl]cholesterol was dissolved in chloroform and evaporated using a rotary evaporator. The dried mixture was hydrated with 200 ॖl of balanced salt solution (137 mm NaCl, 5.4 mm KCl, 10 mmTris-HCl, pH 7.6) containing 100 ॖg of ribozymes. Liposomes were prepared by vortex and extrusion. The liposomes were fused with HVJ that had been inactivated by ultraviolet light; the amount of virus used corresponded to 3 × 104 hemagglutinating units. The HVJ-liposome complexes were separated from unfused HVJ by ultracentrifugation through a 307 (w/w) sucrose layer at 62,800 × g for 90 min. Rat embryonic fibroblasts (Rat-1) transfected with the HTLV-Itax expression plasmid pH2R40M (Rat/Tax cells) have been described previously (17Tanaka A. Takahashi C. Yamaoka S. Nosaka T. Maki M. Hatanaka M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1071-1075Crossref PubMed Scopus (369) Google Scholar). The pH2R40M plasmid contains the SV40 promoter and polyadenylation signal, an R fragment of the HTLV-I long terminal repeat, a neomycin resistance gene, and a HindIII fragment derived from pX, which encodes intact Tax protein. The pH2R40M plasmid expresses the HTLV-I Tax protein, but not the Rex protein. The plasmid (5 ॖg) was mixed with 10 ॖg of Lipofectin (Life Technologies, Inc.) and added to the Rat-1 cells. Transfected cells were selected in medium containing with 800 ॖg/ml G418 sulfate (Geneticin, Life Technologies, Inc.). The resultingtax-transfected Rat-1 cells (Rat/Tax cells) stably express the Tax protein. The Rat/Tax cells were maintained in Dulbecco's modified Eagle's medium supplemented with 107 heat-inactivated fetal calf serum, 4 mml-glutamine, 50 units/ml penicillin, and 50 ॖg/ml streptomycin at 37 °C in a humidified 57 CO2 atmosphere. To determine ribozyme uptake into the cells, 2 × 105 Rat/Tax cells were incubated with 1 ॖm naked FITC-labeled TR, 1 ॖm FITC-labeled TR complexed with HVJ-cationic liposomes, or 1 ॖm FITC-labeled TR complexed with HVJ-anionic liposomes for 24 h. FITC-labeled-rabbit IgG (10 ॖg/ml) was used as a control. The cells were permeabilized with 707 ethanol for 30 min and incubated with phosphate-buffered saline (PBS) containing 250 ॖg/ml propidium iodide (PI) for 15 min at room temperature in the dark. Cells from each group were mounted and analyzed by confocal laser scanning microscopy (Leica True Confocal Scanner 4D, Leica Lasertechnik GmbH, Heidelberg, Germany). The image consisted of 512 × 512 pixels with the FITC detection system. Cells containing FITC-labeled ribozymes were counted by flow cytometry using an EPICS Profile counter (Coulter Electronics Inc., Hialeah, FL). The cut-off value for positive staining by PI was set at 10 on the y axis, and the cut-off value for cells positive for TR was set at 10 on the x axis in the histogram. Rat/Tax cells (2 × 105) were incubated for 4 days without ribozymes or with 1 ॖmnaked TR, 1 ॖm TR complexed with HVJ-cationic liposomes, 1 ॖm RR complexed with HVJ-cationic liposomes, 1 ॖm TC complexed with HVJ-cationic liposomes, 1 ॖm tax antisense ODNs complexed with HVJ-cationic liposomes, or 1 ॖm tax sense ODNs complexed with HVJ-cationic liposomes. Furthermore, cells were treated with 1 ॖm TR complexed with HVJ-cationic or -anionic liposomes diluted by a factor of 1 × 10−1 to 1 × 10−3. The cells were lysed with radioimmune precipitation buffer (25 mm Tris-HCl, pH 7.5, 50 mm NaCl, 0.57 Nonidet P-40, 0.57 sodium deoxycholate, 0.17 SDS, 17 aprotinin, and 1 mm phenylmethylsulfonyl fluoride) at 98 °C. The proteins were fractionated on an 87 SDS-polyacrylamide gel and transferred to an Immobilon P membrane (Nihon Millipore, Ltd., Tokyo, Japan). The membrane was blocked with 37 fat-free milk in PBS for 15 h at 4 °C and incubated for 1 h with anti-HTLV-I Tax monoclonal antibody (mAb) (gift of Prof. M. Hatanaka, Kyoto University) (diluted 1/1000). The membrane then was washed four times with PBS containing 0.17 Tween, followed by incubation with peroxidase-conjugated goat anti-mouse IgG antibody (Cappel Research Products, Durham, NC) (diluted 1/10,000) for 1 h at room temperature. Immunoreactive proteins were visualized by enhanced chemiluminescence using the ECL system (Amersham, Buckinghamshire, UK). Rat/Tax cells (2 × 105) were incubated for 4 days without ribozymes, with 1 ॖm naked TR, 1 ॖm TR complexed with HVJ-anionic liposomes diluted by a factor of 1 × 10−2, or 1 ॖm TR complexed with HVJ-cationic liposomes diluted by a factor of 1 × 10−2. The cells were permeabilized with 707 ethanol for 30 min. After incubation with saturating concentrations of anti-HTLV-I Tax mAb (1:500 dilution) for 30 min at 4 °C, the cells were treated with FITC-labeled anti-mouse IgG (Cappel Research Products) as a second antibody. Control samples were treated with anti-FITC-labeled mouse IgG alone. The expression of Tax protein was quantified by flow cytometry (Coulter Electronics Inc). Rat/Tax cells (3 × 104) grown on glass coverslips for 4 days were treated with or without ribozymes as described above and fixed with 507 acetone, 507 methanol. Autofluorescence was quenched by treatment with 50 mm NH4Cl. The slides were rinsed in PBS containing 17 bovine serum albumin and incubated for 1 h in anti-HTLV-I Tax mAb (diluted 1:300). A tetramethylrhodamine isothiocyanate (TRITC)-conjugated secondary antibody, goat IgG fraction to mouse IgG (Cappel Research Products), was added at a 1:200 dilution and incubated for 30 min at room temperature. Negative control samples were similarly treated but were not exposed to the primary antibody. Confocal laser scanning microscopy (Leica True Confocal Scanner 4D) was used to determine the intracellular localization of both the HTLV-I Tax protein and ribozymes. Each image consisted of 1024 × 1024 pixels with the two-color scanning analysis system. We synthesized two hammerhead ribozymes that were sequence-specific for a site downstream of the AUG start codons of the HTLV-I tax mRNA (TR) or rex mRNA (RR) (Fig. 1 A). These sites had previously been targeted successfully by antisense ODNs (18Kitajima I. Shinohara T. Minor T. Bibbs L. Bilakovics J. Nerenberg M. J. Biol. Chem. 1992; 267: 25881-25888Abstract Full Text PDF PubMed Google Scholar, 20Kitajima I. Kawahara K. Hahyu N. Shin H. Tokioka T. Soejima Y. Tstutsui J. Ozawa M. Shimayama T. Maruyama I. J. Cell Sci. 1996; 109: 609-617PubMed Google Scholar) and thus were expected to be accessible for ribozyme-mediated cleavage. Incubation of the 220-base substrate HTLV-I tax/rex mRNA with RR resulted in the predicted 97-base and 123-base cleavage products, whereas 148-base and 72-base cleavage products were observed following incubation with TR (Fig. 1 B). Both cleavage reactions were dose-dependent and were also dependent on the Mg2+ concentrations (Fig. 1 C). No cleavage products were observed when the mRNA was incubated with the inactive control ribozymes, RC and TC (Fig. 1 B). We previously established a novel and highly efficient method for gene transfer using HVJ-liposomes (11Kaneda Y. Uchida T. Kim J. Ishiura M. Okada Y. Exp. Cell Res. 1987; 173: 56-69Crossref PubMed Scopus (92) Google Scholar, 12Kaneda Y. Iwai K. Uchida T. Science. 1989; 243: 375-379Crossref PubMed Scopus (424) Google Scholar). Gene delivery was even most efficient if the liposomes consisted of cationic rather than non-cationic lipids (13Saeki Y. Matsumoto N. Nakano Y. Mori M. Awai K. Kaneda Y. Hum. Gene Ther. 1997; (in press)PubMed Google Scholar). In the present study, we have compared the gene transfer efficiency of ribozymes in the absence of HVJ-liposomes (naked ribozymes) and in the presence of HVJ-cationic or -anionic liposomes. To evaluate the intracellular distribution of the ribozymes, FITC-labeled ribozymes were visualized by confocal laser scanning microscopy. Cells of control samples containing FITC-conjugated rabbit IgG exhibited no staining (Fig.2 A). Incubation of Rat/Tax cells with naked ribozymes for 24 h resulted in weak staining and spotty distribution of ribozymes in the cytoplasm of 1–37 of the cells (Fig. 2 B). Examination of the cells at a higher magnification demonstrated that the naked ribozymes were distributed diffusely throughout the cytoplasm. TR complexed with HVJ-anionic liposomes showed a significantly improved cellular uptake compared with the naked ribozymes (Fig. 2 C). Most of these ribozymes were retained in the cytoplasm and existed as vesicles in the endosomes or lysosomes (data not shown). Approximately 207 of cells also exhibited staining in their nuclei. In contrast, more than 907 of the Rat/Tax cells incubated with TR in the presence of HVJ-cationic liposomes exhibited cytoplasmic and nuclear fluorescence, which was more intense than in cells treated with anionic liposomes (Fig. 2 D). To quantitate the cellular uptake of TR complexed with HVJ-cationic liposomes, cells containing FITC-labeled ribozymes were counted by flow cytometry. Positive staining was indicated by a signal shift to the right on the x axis, compared with that of a negative control sample stained with mouse IgG (Fig.3, left panels). Rat/Tax cells positive for nuclear staining by PI were located above the cut-off value of 10 on the y axis (Fig. 3, right panels,regions a and b). Cells containing FITC-labeled ribozymes were located to the right of the cut-off value of 10 on thex axis (regions b and d). Region b of the histogram represented double-positive cells, whose proportion we estimated. These analyses indicated that naked TR was present in 57 of the 104 cells after a 24-h incubation (Fig. 3 A), whereas ribozymes complexed with HVJ-anionic liposomes had been taken up into 22.87 of the cells (Fig.3 B). In contrast, ribozymes complexed with HVJ-cationic liposomes demonstrated a significantly increased uptake and were delivered into 96.17 of the cells (Fig. 3 C). Thus, the uptake of ribozymes complexed with HVJ-cationic liposomes was 15–20 times increased compared with naked ribozymes and 4–5 times increased compared with ribozymes complexed with HVJ-anionic liposomes. Rat/Tax cells express high levels of taxmRNA, but no rex mRNA, because the taxcDNA used transfected these cells contained naked 5′-rexsequences (17Tanaka A. Takahashi C. Yamaoka S. Nosaka T. Maki M. Hatanaka M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1071-1075Crossref PubMed Scopus (369) Google Scholar) (Fig. 4 A). Thus, the tax mRNA from Rat/Tax cells could be cleaved by TR but not by RR. We used Western blot analysis to investigate whether the ribozymes introduced into the cells by HVJ-cationic liposome-mediated gene transfer possessed sequence-specific catalytic activity and therefore down-regulated Tax expression. The Tax protein signals were quantified by densitometric scanning. The 40-kDa Tax protein was highly detectable in Rat/Tax cells treated with 1 ॖm naked TR (Fig. 4 B, lane 1). Treatment with 1 ॖm TR complexed with HVJ-cationic liposomes significantly reduced Tax expression by approximately 957, compared with cells treated with 1 ॖm naked TR (Fig.4 B, lane 2). In contrast, Tax protein expression was not affected by treatment with 1 ॖm RR complexed with HVJ-cationic liposomes (Fig. 4 B, lane 3). A reduction of nearly 207 in Tax protein expression was observed in cells treated with 1 ॖm TC complexed with HVJ-cationic liposomes (Fig. 4 B, lane 4). However, this effect of TC may be due to an antisense effect of the flanking TR sequences. Consistent with this hypothesis, a similar reduction in Tax expression of about 207 was also found in cells treated with 1 ॖm tax antisense ODNs (18Kitajima I. Shinohara T. Minor T. Bibbs L. Bilakovics J. Nerenberg M. J. Biol. Chem. 1992; 267: 25881-25888Abstract Full Text PDF PubMed Google Scholar, 20Kitajima I. Kawahara K. Hahyu N. Shin H. Tokioka T. Soejima Y. Tstutsui J. Ozawa M. Shimayama T. Maruyama I. J. Cell Sci. 1996; 109: 609-617PubMed Google Scholar) complexed with HVJ-cationic liposomes (Fig. 4 B, lane 5). In contrast, Tax expression was not reduced after treatment with 1 ॖm tax sense ODNs, complexed with HVJ-cationic liposomes (Fig.4 B, lane 6). We compared the degree of down-regulation of Tax expression obtained when cationic or anionic liposomes that carried equal amounts of TR were used. Under these conditions, TR complexed with HVJ-cationic or HVJ-anionic liposomes produced a similar level of Tax down-regulation (data not shown). To evaluate the ribozyme achieved with each type of HVJ-liposome more precisely, we also compared Tax suppression after ribozyme transfer using diluted HVJ-liposomes. When the Rat/Tax cells treated with 1 ॖm TR complexed with HVJ-cationic liposomes diluted by a factor of 1 × 10−2, a reduction of Tax expression occurred (Fig.5, lanes 2–4). In contrast, 1 ॖm TR complexed with HVJ-anionic liposomes diluted by a factor of 1 × 10−1 showed a decrease in Tax protein expression (Fig. 5, lanes 5–7). Densitometric scanning analysis of a Tax immunoblot demonstrated that Tax signals were approximately 5 times lower in cells incubated with 1 ॖmTR complexed with 1 × 10−2 diluted HVJ-cationic liposomes (Fig. 5, lane 3), compared with equally diluted HVJ-anionic liposomes (Fig. 5, lane 6). We quantified the inhibitory effects of TR on Tax expression by flow cytometry using an anti-Tax mAb as reported previously (20Kitajima I. Kawahara K. Hahyu N. Shin H. Tokioka T. Soejima Y. Tstutsui J. Ozawa M. Shimayama T. Maruyama I. J. Cell Sci. 1996; 109: 609-617PubMed Google Scholar). Rat/Tax cells expressed Tax protein at a high level (Fig.6 A). In cells treated with 1 ॖm naked TR, the level of Tax expression was almost unaffected (Fig. 6 B). Incubation of the cells with 1 ॖm TR complexed with HVJ-anionic liposomes diluted by a factor of 1 × 10−2 (Fig. 6 C), however, considerably down-regulated Tax expression, although to a lesser extent than did 1 ॖm TR complexed with an equal volume of HVJ-cationic liposomes (Fig. 6 D). The intracellular distribution and kinetics of ribozyme activity in the Rat/Tax cells was visualized using FITC-labeled ribozymes. The relationship between the ribozymes and Tax protein was investigated by a two-color confocal laser-fluorescence-microscopy scanning system using FITC-labeled ribozyme, which produces a green signal, and TRITC-labeled Tax protein, visible as an orange signal. A yellow signal indicated the co-localization of ribozyme and Tax protein. All untreated Rat/Tax cells expressed Tax protein both in the cytoplasm and in the nucleus, and the expression level did not differ among individual cells (data not shown). When the cells were incubated with TR complexed with HVJ-cationic liposomes for 4 days, Tax protein expression decreased markedly, resulting in the degradation of Tax protein, as indicated by the loss of orange signals (Fig.7 A). Approximately 57 of the cells did not incorporate TR and still expressed Tax protein (Fig. 7A, arrow). In contrast, following the introduction of RR mediated by HVJ-cationic liposomes, yellow signals were clearly evident in the cytoplasm and nuclei of the Rat/Tax cells (Fig. 7 B). This observation suggests that Tax protein was still highly expressed following the nuclear deposition of RR. The present study evaluated the efficiency of a method of gene transfer using anionic or cationic liposomes complexed with HVJ to introduce hammerhead ribozymes targeted against HTLV-Itax/rex mRNA into living cells. We also investigated the sequence specificity and activity of these ribozymes in the cells. We found that transfer efficiency was higher for ribozymes complexed with HVJ-cationic liposomes rather than with HVJ-anionic liposomes. The ribozymes specifically cleaved the HTLV-I tax/rex target mRNA, and TR suppressed Tax protein expression in the cells. The ultimate goal of gene therapy for HTLV-I infections is the inactivation of viral genes in the infected cells. Tax protein has been suggested to play a role in tumorigenesis by modulating the expression of cellular genes (26Yoshida M. Roman G.C. Vernant J.-C. Osame M. HTLV-I and the Nervous System. Alan R. Liss, Inc., New York1989: 19-29Google Scholar). We previously reported t
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