Bioconjupte Chem. 1993, 4, 448-454
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Efficient Transformation of Mammalian Cells Using DNA Interpolyelectrolyte Complexes with Carbon Chain Polycations Alexander V. Kabanov,* Irina V. Astafieva, Irina V. Maksimova,+ Eugenii M. Lukanidin,+J Georgii P. Georgiev,+ and Victor A. Kabanovs Moscow Institute of Biotechnology, Inc., and Department of Biopolymer Chemistry, Russian Research Center of Molecular Diagnostics and Therapy, Simpheropolsky Boulevard 8, Moscow 113149, Russia, Institute of Gene Biology, ul. Vavilova 34/5, Moscow 117334 GSP-1, Russia, and Department of Polymer Chemistry, Faculty of Chemistry, M. V. Lomonosov Moscow State University, Vorobievi Gory, Moscow 119899 GSP, Russia. Received April 26, 1993”
A new method for mammalian cell transformation is proposed which is based on incorporation of plasmids into interpolyelectrolyte complexes (IPECs) with carbon chain polycations. The method is illustrated by examples of pRSV CAT and p@-Galplasmid IPECs with poly(N-ethyl-4-vinylpyridinium bromide) (C2PVP) and poly(N-ethyl-4-vinylpyridinium)-poly(N-cetyl-4-vinylpyridinium) bromides random copolymer (ClSpVP). These IPECs are produced spontaneously due to formation of a cooperative system of interchain electrostatic bonds after mixing DNA and polycation solutions. The interaction of IPEC with normal mouse fibroblasts NIH 3T3, human T-lymphoma “Jurkat”, and Mardin Darby canine kidney cells has been studied. The data obtained has revealed that plasmid incorporation into IPECs significantly enhances both DNA adsorption on the plasma membrane and DNA uptake into a cell. The i n vitro transformation of NIH 3T3 cells was monitored by a standard cloramphenicol acetyltransferase (CAT) assay (pRSV CAT plasmid) and by detection of @-galactosidase(@-Gal)expression using 4-methylumbeliferril @-D-galactopyranosideas a substrate (p@-Galplasmid). In both cases it has been proved that IPEC-incorporated plasmids possess an ability for efficient cell transformation. The transforming activity of IPECs depends on their composition and polycation chemical structure. Under optimal conditions the efficiency of cell transformation with IPECs is several fold higher than that observed during standard calcium phosphate precipitation. The mechanism of the phenomenon observed is discussed. According to this mechanism polycations form positively charged loops fixed on DNA molecules. These loops interact with negatively charged lipids on plasma membranes, which probably induces effective endocytosis and subsequent cell transformation.
INTRODUCTION Targeting of foreign nucleic acids into intact cells is the basis of many key genetic engineering methods ( I ) . At present there exist several methods for introducing DNA into cells, the most common of them being precipitation with calcium phosphate (2) or with DEAE-dextran’ (3),
* Address all correspondence to A. V. Kabanov, Moscow Institute of Biotechnology, 550 Rue Sherbrooke Quest, Bureau 1205, Montreal, QC, Canada, H3AlB9. + Institute of Gene Biology. Present address: The Fibiger Institute, Strandboulevarden 49, opg. 7.1, 3,s. DK-2100 Copenhagen, Denmark. f Moscow State Univeristy. Abstract published in Advance ACS Abstracts, October 1, 1993. 1 Abbreviations used B-Gal, &galactosidase; CzPVP, poly(N-ethyl-4-vinylpyridiniumbromide);ClsPVP, poly(N-ethyl-4vinylpyridinium)-poly(N-cetyl-4-vinylpyridinium)bromides random copolymer; CAT, chloramphenicol acetyltransferase; Con A, concanavalin A; DEAE-dextran, [2-(diethylamino)ethyl]dextran; DMEM, Dulbecco’s modified Eagle’s medium; FACS, fluorescence-activated cell sorter; p, IPEC composition, namely the basic mole ratio [polycation]/[DNA]in the system; HBS, HEPES-buffered saline;HEPES,N-(2-hydroxyethyl)piperazineN’-(2-ethanesulfonicacid);IPEC; interpolyelectrolytecomplex; MDCK, Mardin Darby canine kidney (cells);Pw,weight-average degree of polymerization; PANa, sodium poly(acry1ate); PBS, phosphate-bufferedsaline;PVP, poly(viny1pyridine);TLC,thinlayer chromatography; Tris, 2-amino-2-(hydroxymethyl)-l,3propanediol; Triton X-100, a-[4-(1,1,3,3-tetramethylbutyl)phenyl]-w-hydroxypoly(oxy-1,2-ethanediyl).
electroporation (4),microinjection (5),and incorporation of DNA into reconstructed virus coats (6) or liposomes (7).
Despite the great variety of these methods, the search for new ways of transforming animal, plant, and prokaryotic cells continues. On the one hand, there is need to enhance the efficiency of transformation in comparison with that achieved by using traditional approaches, and that can be applied only to a limited number of cell lines (8). On the other hand, traditional approaches seem to be ineffective for introducing RNA molecules into cells (9). And finally, most of these approaches cannot be used for genetic transformation i n vivo (10). Recently, a new approach has been suggested based on incorporation of nucleic acids into soluble interpolyelectrolyte complexes (IPECs) with polycations (11-13). In particular, Wu et al. (11) suggested conjugation of a polycation with a molecule (“vector”)capable of receptormediated endocytosis into a cell. The mixing of this conjugate with DNA polyanion resulted in formation of IPEC carrying a ligand which provided for receptormediated uptake of IPEC and cell transformation. Wu et al. used asialoglycoprotein-poly(L-lysine) conjugate for transformation of receptor-positive HepG2 hepatoma cells (11)as well as for DNA-targeted delivery into liver and hepatocyte transformation i n vivo (10, 14). In further studies transferrin (8, 15, 16) and insulin (17)were used as vectors instead of asialoglycoprotein for transforming various eukariotic cells i n vitro. On the basis of the works by Wu e t al. (10,14), Trubetskoy et al. (18) developed a system for gene targeting in mouse lung endothelian cells
1043-1802l93l2904-0448~04.QQlQ @ 1993 Amerlcan
Chemical Society
DNA Polycomplexes for Cell Transformation
using a poly(i-lysine) conjugate with mAb to lung-surface antigens as a DNA carrier. Meanwhile, we demonstrated that DNA incorporation into IPEC with vector-free carbochain polycations (namely quaternized PVPs) significantly enhanced the efficiency of transformation of Bacillus subtilis cells (12,191. This phenomenon was explained (19) by hydrophobization of DNA molecules upon their incorporation in IPEC, which intensified interaction of the latter with the cellular membrane. A similar approach based onDNA complexion with lipospermines was used by Behr et a1. for transforming mammalian cells (13). In this work we study transformation of mammalian cells using DNA IPECs with quaternized PVPs. EXPERIMENTAL PROCEDURES
Synthetic Polymers. PVP was obtained by radical polymerization of freshly rectified 4-vinylpyridine (boiling temperature of 65 "C at 15 mmHg, n z o =~ 1.5520) using the modified method (20). The polymerization reaction in 25% monomer solution in methanol was initiated by the azodinitrile of bisisobutiric acid and conducted under an argon atmosphere at 65 "C during 40 h. The polymer obtained was precipitated into ether and dried under vacuum. The break up fractionation of PVP was performed by precipitation of 1-5 % methanol solution of the polymer into ethyl acetate. The obtained liquid PVP layer was separated, washed, dissolved in methanol, and precipitated into ether. The molecular mass of the high molecular PVP fractions was determined using quasielastic light scattering. The molecular mass of low molecular (oligomeric)PVP fractions was determined by ebulioscopy in chloroform. CzPVPs (Pw= 18and 500) and c16PvP (Pw= 400) were obtained by alkylation of corresponding PVP fractions with cetyl and/or ethyl bromides according to the procedure previously described (21). The degree of PVP alkylation was determined by IR spectroscopy (21). According to these data, CzPVPs contained from 90 to 95 % alkylated units and c16PvP contained 3 % and 90 % cetylated and ethylated units, respectively. The polymers obtained were purified by dialysis and gel filtration on Biogel P2, lyophilized, and then dissolved in doubly distilled water with a partial conductivity of 0.06 ps/cm (purified by a "Milli-Q" device, Millipore). The concentrated (from 1to 3 basic mmol/L) stock solutions of CzPVPs and cl6PvP were sterilized in an autoclave for 30 min (0.5 atm, 100 "C) and stored under sterile conditions. The polymers concentration in the solutions obtained were controlled spectrophotometrically by measuring the absorbency of N-alkylpyridinium bromide units at 280 nm (the coefficient of molar absorption of the unit equals 3500 opt units/mol). Cell Lines and Culture Conditions. NIH 3T3 cells were grown at 37 "C, under a 5% COz atmosphere as a monolayer stationary culture in DMEM, containing 2 mM glutamine and 10% fetal calf serum. To obtain the cell suspension the exponentially growing NIH 3T3 cells were harvested by trypsin treatment and then grown for at least three passages (37 "C, 5% COZ). Mardin Darby canine kidney (MDCK) cells were grown (37 "C, 5% COZ)as a monolayer stationary culture in 199 medium containing 10 mM HEPES, 0.075% sodium bicarbonate, 10% fetal calf serum, and 200 pg/mL gentamycin. Jurkat cells were grown (37 "C, 5% COZ)as a suspension culture in RPMI1640 medium containing 2 mM glutamine and 10% fetal calf serum. Cell Viability. The cells were incubated in serum-free medium for 1h at 37 "C with the polycations or IPECs.
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The cells were then washed and incubated for 48 h in the fresh medium containing 10% fetal calf serum at 37 "C. In the course of incubation (24 h), the medium was substituted for the fresh one. The amount of living cells was determined via FACS analysis using the propidium iodide fluorescein diacetate staining (22). Plasmids. pRsV CAT and p@-Galplasmids were kindly presented by Dr. E. Tulchinsky (V.A. Engelgardt Institute of Molecular Biology,Russian Academy of Sciences).They were propagated and purified by standard techniques (23). Plasmid pRSV CAT (24)was composed of the ampicillinresistant cistron and the origin of replication was from plasmid pBR322 by joining to a hybrid eukaryotic transcription unit. The transcription unit in this plasmid was composed of the Rous sarcoma virus long terminal repeat (Rous LTR) promoter, the driving expression of Escherichia coli CAT encoding sequence, and the simian virus 40 (SV40)mRNA processing signals (including the small4 intron and early region polyadenilation site). Plasmid p@Gal was composed of the ampicillin-resistant cistron and the origin of replication from plasmid pUC19 by joining to a hybrid eukaryotic transcription unit, which contained the E. coli 0-Gal encoding sequence. DNA Radiolabeling. DNA was labeled with [ C U - ~ ~ P I dCTP according to a standard protocol for nick translation reactions using an Amersham nick translation kit. The specific radioactivity of labeled DNA equaled 3.70106cpm/ rg. Polycomplex Formation. The IPECs were obtained by simple mixing of polycation and DNA solutions. To this end, from 0.5 to 50 pL of polycation stock solution was added to 5 pL of 0.2 pg/pL DNA solution in PBS or HBS (pH 7.4). According to the data previously reported (19), rapid (minutes) IPEC formation took place under these conditions. The system obtained was incubated for 1 h at room temperature, filtered using 0.22-pm filters, and then used in further experiments. An independent experiment revealed that IPEC filtration did not result in the loss of polycomplex components. The basic molar [polycation]/ [DNA] ratio is conditionally termed below as IPEC composition (rp). DNA Binding and Internalization in Cells. Suspended Jurkat (2.109 and 3T3 (5.105) cells or monolayers of 3T3 and MDCK cells in 90-mm plates were washed with cold (4 "C) serum-free medium and then placed in 1 mL of serum-free medium containing 20 mM HEPES (pH 7.4). From 0.02 to 0.6 pg of radiolabeled DNA or ita IPECs with CZPVPin PBS (pH 7.4) or HBS (pH 7.4) were added to the cells. After 1.5-h incubation at 4 or 37 "C, the cells were washed from three to four times with cold (4 "C) PBS and then treated according to one of the following procedures for determination of the amounts of bound (A) or internalized DNA (B-D). (A)The cells were lysed a t 0 "C using 0.1 % Triton X-100 solution in 1 mM Tris-HC1 buffer (pH 7.41, and [32Pl radioactivity in the lysate was measured in a toluene scincillator using a Rackbeta-I1 @-counter. The concentration of cell protein in the lysate samples was determined by the Bradford method (25). (B) The cells were placed in 1mL of 50 mM Tris-HC1 buffer (pH 5.0) containing 4.2 mM MgC1, 0.1% DTT, and 6 pL of 1mg/mL DNAse I solution. After 1hour incubation at 4 "C, the cells were carefully washed and lysed. The DNA radioactivity and concentration of cell protein were determined as described in A. (C) The cells were incubated for 1h in 1mL of cold (4 "C) PBS (pH 7.8) containing from 20 to 30 pL of 10 mM
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PANa solution and then washed and treated according to the procedure described in B. (D) The cells were treated for 6 min at 4 "C with 0.2 M acetic acid (pH 2.5) containing 0.15 M NaCl and then washed and lysed. The DNA radioactivity and concentration of cell protein in the lysate were determined as described in A. For comparison, the binding and internalization of the same amounts of DNA during calcium phosphate precipitation were also studied using the protocol described below. Cell Transformation Using IPECs. In order to produce cell monolayers, suspended NIH 3T3 cells were seeded at 5 ~ 1 per 0 ~ 90-mm plate and incubated overnight in 10 mL of growth medium. Twenty-four hours before the transformation the serum-containing medium was substituted for the serum-free one. One hour before the transformation the suspended NIH 3T3 cells (%lo5to 109 were washed and resuspended in 0.5 mL of the serum-free medium. Two to 5 mL (cellmonolayers)or 0.5 mL (cellsuspension) of the serum-free medium containing 2-16 pg of DNA or its IPECs of varying composition was added dropwise to the plate of cells or 0.5 mL of the cell suspension. The mixtures were gently agitated and incubated for 2 h at 37 "C under 5% C02. Then the cells were washed five or six times with the fresh media (cells in suspension were replated to dishes) and incubated in DMEM containing 10% fetal calf serum for 48 h at 37 "C under 5% CO2. In the course of incubation (16h) the medium was substituted for the fresh one. After incubation, the cells were washed twice in PBS, trypsinized, and washed twice in PBS, and CAT or @-Galactivities were determined. Cell Transformation Using Calcium Phosphate Precipitation. The calcium phosphate transformation was performed using the optimized protocol described in ref 26. To this end 2-16 pg of plasmid DNA was mixed with 0.5 mL of 0.25 M CaC12. This mixture was slowly added dropwise to 0.5 mL of 2XHBS (pH 7.1) with continuous vortexing (22-25 "C). The obtained slightly opaque solution was incubated for 10 min at room temperature and then added dropwise to the plate of cells or to the suspended cells. The mixture was swirled gently and incubated for 24 h at 37 "C under 5% C02. The medium was removed, and the cells were rinsed twice with growth medium (cells in suspension were replated to dishes) and incubated in DMEM containing 10% fetal calf serum for an additional 24 h at 37 "C under 5 % C02. After incubation, the cells were washed and trypsinized, and CAT or @-Galactivities were determined. The above protocol is optimized (pH, sample volumes, temperature) for NIH 3T3 cell transformation with p@-Galplasmid. CAT Assay. The CAT activity in the transformed cells was determined by the method of Gorman et al. (27). Briefly, the cells were suspended in 100 pL of 0.2 M TrisHC1 (pH 7.4),lysed by several freeze/thaw cycles, incubated for 10 min at 65 "C for inactivation of diacetylases, and centrifuged in an Eppendorf centrifuge at 3000 rpm. A 50-pL portion of supernatant was then mixed with 50 pL of Tris-HC1 buffer containing 20 pCi/mL V4C1chloramphenicol (53 mCi/mmol). The reaction was initiated by adding 20 pL of 4 mM Con A solution. After 1-h incubation, cloramphenicol and the products of its enzymatic hydrolysis were extracted with ethyl acetate, analyzed by TLC, and autoradiographed. Each transformation experiment using pRSV CAT plasmid (IPEC or calcium phosphate precipitation) was performed in duplicate (two samples for each experimental point) and
Table I. Effect of Polycations on NIH 3T3 Cell Viability polycation concentration viability after polycation (basic mmol/L) incubationa (%) 75.0 control (without polycation) 75 80.4 CzPVP (Pw= 18) 16 80.7 CZPW (Pw= 500) C16pVP (Pw= 400) 850 47.9 50 75.6 a 3T3 NIH cell Suspension was incubated with polycations for 1 h at 37 "C. The cells were then washed and incubated for 48 h at
37 "C as described in Experimental Procedures.
repeated independently at least four times. The results of the typical experiments are presented below. &Gal Assay. The cells were suspended and lysed by several freeze/thaw cycles as described above for determination of CAT activity. However, in this case the extract was not incubated at 65 "C. After the extract centrifugation, 50 pL of supernatant were mixed with 50 pL of 1 mM 4-methylumbeliferril P-D-galactopyranosidesolution in 0.1 M phosphate buffer (pH 7.4) and then incubated for 20 min at 37 "C. The reaction was terminated by adding 50 gL of 0.4 M glycine buffer (pH 10.5). The formation of methylbelliferon (bx = 365; A,, = 450) was monitored at 37 "C by a Hitachi F 4000 spectrofluorimeter. RESULTS
Effect of Polycations on Cell Viability. The effects of C2PVP and c16PVP on cell viability were studied. Briefly the cells were incubated with the polycations, washed, and then incubated again in the fresh media under conditions similar to those used in the transfection experiments. After incubation the cell viability was determined by a standard FACS technique (22). Table I shows that, in the case of NIH 3T3 cells, the polycations did not affect cell viability at concentrations that exceeded by manifold those used during transfection. A similar result was obtained when corresponding IPECs were used instead of free polycations (data not presented in the table). Therefore, unlike calcium phosphate treatment, which usually leads to a loss of from 50 to 90% of the cells, the transfection procedure proposed in this paper does not possess a cytotoxic effect. A Method for Separate Determination of DNA Binding and Uptake. The interaction of free and IPECincorporated plasmids with the cells was studied. To this end, the cells (3T3, MDCK, Jurkat) were incubated with [32P]-labeled pRSV CAT plasmid (or its IPEC with C2PVP) at 4 or 37 "C, washed, and then treated according to one of the procedures (A-D) (described in Experimental Procedures), permitting separate determination of DNA binding and uptake. Here we illustrate this approach by examples of plasmid (or IPEC) interaction with NIH 3T3 suspension (Table 11). The total amount of the cell-bound DNA was determined by measuring DNA radioactivity in the cell lysate (procedure A). In order to remove DNA molecules adsorbed on the cell surface and thus to determine the amount of internalized nucleic acid, the cells were first treated with DNAse I and then lysed (procedure B). Such treatment resulted in complete removal of cell-bound DNA in cases when the cells were incubated with the free plasmid (Table 11). This indicates that the free DNA adsorbs on the cell surface only but does not internalize within the cells. After calcium phosphate precipitation, a significant part of cell-bound DNA was not accessible to DNAse digestion. This part corresponded to DNA internalized into the cells during the precipitation procedure. The part of DNA
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DNA Polycomplexes for Cell Transformation
Table 11. Interaction of Free or IPEC-Incorporated pRSV CAT Plasmid with NIH 3T3 Cell Suspension
system studieda free DNA calcium phosphate precipitation IPEC
cell-boundDNA (ng/mg cell protei&
temp (OC)
A
B
C
D
37 37
7(i2) 20(i2)
2(il) 11(*3)
