To understand the relative contribution of viral
receptor expression and cell proliferation in retroviral gene transfer,
we created human hepatocyte-derived HuH-7.MCAT-1 cell lines. These cells constitutively express the murine ecotropic retroviral receptor MCAT-1 without changes in morphology or proliferation states. The
MCAT-1 receptor is also a cationic amino acid transporter, and the
HuH-7.MCAT-1.7 cells showed increased Vmax of
uptake and steady-state accumulation of the cationic amino acids
L-arginine and L-lysine. In HuH-7.MCAT-1 cells,
L-arginine uptake was significantly up-regulated by
norepinephrine and dexamethasone, and hepatocyte growth factor also
increased L-arginine uptake along with cellular DNA
synthesis. Gene transfer was also markedly increased in HuH-7.MCAT-1.7 cells incubated with an ecotropic LacZ retrovirus, and this further increased with hormones and hepatocyte growth factor. To define whether
viral receptor up-regulation by itself increased gene transfer, cell
cycling was inhibited by a recombinant adenovirus expressing the Mad
transcription factor (AdMad), which is a dominant-negative c-Myc
regulator. This restricted cells in G0/G1,
without attenuating MCAT-1 activity, as shown by flow cytometry and
L-arginine uptake analysis, respectively. When
asynchronously cycling HuH-7.MCAT-1.7 cells were first infected with
the AdMad virus and then exposed to the ecotropic LacZ virus, gene
transfer was virtually abolished. The data indicate that while
up-regulation of viral receptors can greatly enhance retrovirally
mediated gene transfer, DNA synthesis remains an absolute requirement
for hepatic gene therapy with this approach.
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INTRODUCTION |
Retroviral gene transfer vectors are desirable because of their
safety and integration into the host genome, which permits permanent
expression of introduced genes. However, proviral integrations in the
host genome require cell proliferation and even ongoing mitosis (1). On
the other hand, the frequency of cell proliferation-related events
differs among cell types and may govern why cycling cells incorporate
retroviruses far more avidly than quiescent cells (2). Additional
factors determining retroviral gene transfer include the presence or
absence of specific receptors that determine viral entry into cells (3,
4). The specificity of retroviral infection is exhibited at two levels:
species-specific and cell type-specific. The former is determined by
differences in the viral envelope, and the latter by the presence of
specific cell membrane receptors, which make cell types susceptible to
infection with a given retrovirus. Alternative vectors, particularly
adenovirus, which in contrast with retroviruses can be produced in
extremely high titers, suffer from episomal deposition of introduced
genes, leading eventually to gene losses, as well as from deleterious host immune responses preventing repeated virus administration (5).
Ecotropic retroviruses utilize the murine cationic amino acid
transporter (MCAT-1)1 as
their cellular receptor, which has recently been characterized (6-11).
MCAT-1 possesses 622 residues and 14 transmembrane-spanning domains;
transports the cationic amino acids arginine, lysine, and ornithine;
and belongs to the so-called y+ transporter system (see
Refs. 11-13 for review). Transfection experiments showed that MCAT-1
expression makes nonpermissive cells susceptible to ecotropic
retroviruses (4). Although the MCAT-1 mRNA is expressed under basal
conditions in many adult organs, including the brain, intestine,
stomach, bone marrow, and spleen, this is not so in the normal adult
liver (4, 11). However, proliferating liver cells do express MCAT-1, as
shown by studies in the newborn rat, partial hepatectomy-induced liver regeneration, and cultured primary hepatocytes undergoing DNA synthesis
(4, 11). Indeed, a number of studies showed that retrovirally mediated
gene transfer is increased under similar conditions (2, 10, 14, 15).
Therefore, it has been unclear as to the individual contribution of
viral receptor expression and DNA synthesis in retroviral gene
transfer.
To develop a suitable system for dissociating retroviral receptor
expression and cellular DNA synthesis, we created novel hepatic cell
lines capable of constitutively expressing the MCAT-1 retroviral
receptor. The task was facilitated by using the established HuH-7 cell
line, which was originally derived from a human hepatocellular carcinoma (16). HuH-7 cells are resistant to ecotropic retroviruses because appropriate receptors are lacking. A number of cell clones were
stably transfected with an MCAT-1 cDNA, and the model was verified
for MCAT-1 expression by using L-arginine uptake as a reporter, including testing hormonal and growth factor regulation of
MCAT-1 activity. To suppress cell cycling without interfering with
MCAT-1 expression, we overexpressed the Mad transcription factor, which
preferentially binds to Max and serves to antagonize c-Myc activity in
cells (17). Use of these systems allowed us to investigate whether
overexpression of viral receptors could lead to greater retroviral gene
transfer, whether DNA synthesis was an absolute requirement for
proviral integrations in the setting of receptor overexpression, and
whether a combination of receptor overexpression and cellular DNA
synthesis would be most effective in retroviral gene transfer.
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EXPERIMENTAL PROCEDURES |
Plasmids, Cell Culture, and Cell Selection--
The pJET plasmid
(18) was digested with BamHI and EcoRI to isolate
MCAT-1 cDNA (2.279-kilobase pair fragment), which was subcloned
into the pGEM3Zf(+) plasmid (Promega, Madison, WI) between the
BamHI and EcoRI sites (pGEM3ZMCAT-1 plasmid). The
MCAT-1 cDNA was then cut from the pGEM3ZMCAT-1 plasmid by
HindIII and EcoRI and subcloned into the
multicloning sites between the HindIII and EcoRI
positions of the eukaryotic expression vector pcDNA3 containing the
cytomegalovirus (CMV) promoter/enhancer and the 231-base pair-long
polyadenylation sequence from the bovine growth hormone gene
(Invitrogen, San Diego, CA). In the final pcDNA3MCAT-1 plasmid, the
MCAT-1 cDNA retained the original BamHI site of the pJET
plasmid. This cloning strategy allowed us to use the CMV promoter and
polyadenylation site of the pcDNA3 plasmid with no further
manipulations. The pcDNA3MCAT-1 plasmid coexpressed the neoR gene under the control of the SV40
promoter/enhancer and allowed for cell selections. The cells were
cultured in RPMI 1640 medium containing 10% fetal bovine serum (Gemini
Biochemicals Inc., Calabasas, CA), 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.). The plasmid
pcDNA3MCAT-1 was transfected via liposomes into HuH-7 cells, and
resistant cells were selected with 400 µg/ml GeneticinTM (Life
Technologies, Inc.). After 4 weeks in GeneticinTM, cell clones were
screened for MCAT-1 cDNA integration and mRNA expression as
well as susceptibility to infection by an ectotropic retrovirus. Based
upon these studies, including increased retroviral gene transfer, one
such clone (designated HuH-7.MCAT-1.7) was used for all subsequent
experiments, and this was recloned by limiting dilutions to ensure
cellular homogeneity. In some experiments, primary rat hepatocytes
isolated by previously described collagenase perfusion methods were
also included (19). The viability of primary rat hepatocytes, tested by
trypan blue dye exclusion, was 85-90%, and 2.5 × 104 cells/cm2 were cultured in RPMI 1640 medium
containing 10% fetal bovine serum and antibiotics for 24 h on
dishes coated with rat tail collagen. The NIH 3T3 mouse fibroblasts
were originally from the American Type Culture Collection (Rockville,
MD).
For hormonal induction experiments, 10
5 to
10
7 M dexamethasone, 5-20 µg/ml insulin,
10
4 to 10
6 M norepinephrine, or
20 ng/ml recombinant human hepatocyte growth factor (HGF) was used. The
cells were cultured both with and without arginine in the culture
medium for 24 h before exposure for 2 h to arginine-free
medium containing L-[3H]arginine for
analyzing steady state intracellular accumulation (see below).
DNA Synthesis and Cell
Proliferation--
[3H]Thymidine incorporation into
trichloroacetic acid-precipitable DNA was measured as described
previously (19). Cells were incubated with 3 µCi of
[3H]thymidine/ml of medium (specific activity of 67 Ci/mmol; ICN Inc., Irvine, CA) for 1 h, washed with ice-cold
phosphate-buffered saline (PBS) (pH 7.4), lysed in 0.33 M
sodium hydroxide, and precipitated with 1.2 M hydrochloric
acid and 40% trichloroacetic acid. The DNA was pelleted in a
microcentrifuge and redissolved in sodium hydroxide. In aliquots,
either the radioactivity was counted in a scintillation counter, or DNA
content was measured by a sensitive microfluorometric assay (19). The
number of cells was manually determined with a Neubauer hemocytometer.
For flow cytometry, cells were released by 0.05% trypsin and 0.53 mM EDTA and washed once with Earle's balanced salt
solution containing 1.8 mM CaCl2, 5.3 mM KCl, 0.8 mM MgSO4, 117 mM NaCl2, 1 mM
NaH2PO4, and 5.6 mM
D-glucose (pH 7.4) (Life Technologies, Inc.). The cellular DNA was stained with 50 µg/ml propidium iodide for 10 min using hypotonic shock with 0.1% sodium citrate as described (19), followed
by analysis with a FACScan using Lysis II software (Becton Dickinson
Advanced Cellular Biology, San Jose, CA). All experiments were
performed in at least triplicate and repeated several times.
Transgene Analysis--
Total cellular RNA and genomic DNA from
the parental HuH-7 and HuH-7.MCAT-1 cells were isolated by a
single-step procedure as described (20). Before electrophoresis, DNA
samples (10 µg each) were digested with BamHI and
EcoRI restriction endonucleases (Promega), electrophoresed
on a 1% agarose gel, transblotted, and UV-cross-linked to Hybond-NTM
membranes (Amersham Pharmacia Biotech). Fifteen µg of RNA/sample was
electrophoresed on 1.2% agarose gels containing 0.6 M
formaldehyde, transblotted, and UV-cross-linked to Hybond-NTM
membranes. Equivalent RNA loading was analyzed by ethidium bromide
staining. The blots were prehybridized for 8 h and hybridized for
16 h with a full-length MCAT-1 cDNA at 42 °C as per the
manufacturer. The purified MCAT-1 cDNA insert was labeled with
[
-32P]dCTP by random primer extension to 2-5 × 108 cpm/µg of DNA using a commercial kit (Amersham
Pharmacia Biotech) (21). The transblots were washed under stringent
conditions with a final wash using 0.1 × SSPE at 65 °C (1 × SSPE = 0.18 M NaCl, 0.01 M sodium
phosphate (pH 7.7), and 0.001 M Na2EDTA). Autoradiography was at
70 °C with X-Omat AR film (Eastman Kodak Co.). Ribonuclease protection assays were performed with the HybSpeedTM ribonuclease protection assay kit (Ambion Inc., Austin, TX). The [32P]UTP-labeled riboprobes were produced using a
MAXIscriptTM in vitro transcription kit (Ambion Inc.). A
720-base pair MCAT-1 riboprobe was synthesized by T7 RNA polymerase
with linearized plasmid pGEM3ZMCAT-1 as the template. A 250-base pair
internal control riboprobe was prepared with the SP6 RNA polymerase
using a linearized pTRI mouse
-actin cDNA as the template.
Fifteen µg of RNA/sample was mixed with 8 × 104 cpm
of [32P]UTP-labeled riboprobe and hybridized at 68 °C
for 1 h; the single-stranded unhybridized riboprobe molecules were
subsequently degraded with RNase A/T1 at 37 °C for 30 min. After
precipitation, the samples were loaded on either denaturing 4%
polyacrylamide gels or nylon membranes by slot blotting.
Amino Acid Transport Assays--
The assay using Earle's
balanced salt solution was based on the method initially described by
Gazzola et al. (22).
L-[2,3,4,5-3H]Arginine (64 Ci/mmol),
L-[U-14C]proline (>250 mCi/mmol; Amersham
Pharmacia Biotech), and L-[4,5-3H]lysine
(80-110 Ci/mmol; Sigma) were obtained commercially. After culturing
1 × 105 cells in RPMI 1640 medium for 24 h in
24-well dishes, the medium was switched, and cells were incubated for
1 h in amino acid-free Dulbecco's modified essential medium (Life
Technologies, Inc.) containing 10% dialyzed fetal bovine serum. Cells
were then washed with amino acid-free Earle's balanced salt solution
and incubated with Earle's balanced salt solution containing various
amounts of unlabeled arginine or proline and a constant amount of
[3H]arginine, [3H]lysine, or
[14C]proline (~1 × 106 cpm). Amino
acid uptake was terminated by adding ice-cold PBS containing 0.1%
bovine serum albumin, and cells were transferred immediately to ice.
Cells were washed three times with ice-cold PBS/bovine serum albumin
and extracted with 200 µl of 5% trichloroacetic acid, and activity
in the soluble phase was counted in a liquid scintillation counter. The
extent of nonspecifically associated [3H]arginine,
[3H]lysine, or [14C]proline was estimated
by incubating cells at 4 °C, and this value was subtracted as an
uptake blank from each sample. The kinetic constants
Km and Vmax were determined
for the initial uptake of L-[3H]arginine,
[3H]lysine, and [U-14C]proline in the
presence of Na+. After depletion of amino acids for 1 h, the initial reaction velocities were estimated from a 60-s
incubation over concentrations ranging between 0.02 and 1.00 mM. The protein concentration of each sample was measured
by the Bio-Rad assay after dissolving 5% trichloroacetic
acid-insoluble phase in 200 µl of 0.1 N sodium hydroxide
(Bio-Rad). For demonstrating steady-state accumulation of the
reporters, 1 × 105 cells were incubated in amino
acid-free Dulbecco's modified essential medium containing 10%
dialyzed fetal bovine serum and either 0.02 or 0.8 mM
unlabeled arginine plus [3H]arginine for 2 h, and
incorporation was analyzed as described above. The amino acid uptake in
individual cultures was standardized to the protein content and fit by
least squares to the Michaelis-Menten equation. The least-square
fitting analysis provided computer-derived Km and
Vmax estimates ± S.E. of the estimate.
Viruses--
Recombinant adenoviruses were grown in
E1a-transformed 293 embryonic kidney cells; purified with two rounds of
cesium chloride gradient ultracentrifugation; and dialyzed in 10 mM Tris-HCl (pH 7.4), 1 mM MgCl2,
and 10% glycerol (23). The adenoviral titer was determined by
measuring optical density at 260 nm. For infection, cells were
incubated with medium containing adenoviruses at a multiplicity of
infection (m.o.i.) of 20 for 2 h. The AdMad virus, which expresses
the Mad protein, was used to suppress cell cycling. The Ad
gal virus,
which expresses Escherichia coli LacZ, was used as a control
to exclude nonspecific changes due to adenoviral proteins, and this
virus was provided by the Cell Culture and Genetic Engineering Core of
the Liver Research Center at the Albert Einstein College of Medicine.
An additional control adenovirus was generated by inserting antisense
hepatitis B virus sequences, using the EcoRI/XbaI
fragment from the pCP10 hepatitis B virus plasmid, map positions 3182 to 2143 (24), into the pAdBglII plasmid using the pJM17 system,
obtained with the permission of Dr. M. Imperiale (University of
Michigan) and Dr. F. L. Graham (McMaster University),
respectively, from Dr. M. Horwitz at the Albert Einstein College of
Medicine. After digestion of the AdBglII plasmid with BglII,
the antisense hepatitis B virus sequences previously ligated into an
expression cassette containing the immediate-early CMV promoter and
enhancer and the SV40 small T-antigen splice and SV40 polyadenylation
sites were cloned. The antisense hepatitis B virus adenovirus was
produced by LipofectinTM-mediated transfection of the modified pAdBglII
and pJM plasmids into E1a-transformed 293 embryonic kidney cells. The
transfected 293 cells were cultured for 10-12 days until cytopathic
effect became apparent, and cells were lysed by three freeze-thaw
cycles. The recombinant adenovirus was grown and purified as described
above.
Ecotropic and amphotropic retroviruses expressing E. coli
LacZ were used for demonstrating gene transfer. The
-CRE-nls-LacZ 30 producer cells were originally developed by Somatix Corp. (Los Angeles,
CA) and provided by the Cell Culture and Genetic Engineering Core of
the Marion Bessin Liver Research Center. The producer cells were
maintained in
-minimal essential medium (Life Technologies, Inc.)
supplemented with 10% calf serum and antibiotics. The culture supernatant was harvested after overnight incubation of cells with
fresh medium and passed through a 0.45-µm filter to remove debris.
Target cells were infected with retroviruses in serum-free Dulbecco's
modified essential medium containing 8 µg/ml Polybrene for 2 h
and cultured for an additional 72 h before fixation in 0.5%
glutaraldehyde in PBS for 10 min. After washing with PBS, fixed cells
were incubated overnight with 1 mg/ml 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside in 5 mM potassium
ferricyanide, 5 mM potassium ferrocyanide, and 2 mM magnesium chloride. For determining the viral m.o.i., a
constant number of NIH 3T3 cells (1 × 105) were
incubated with serially diluted retrovirus medium, and the number of
blue-stained cells was counted in random areas. For some experiments,
retroviruses were concentrated by precipitation with 5 mM
calcium chloride as described (25).
Statistical Methods--
Data are expressed as means ± S.E. Data were analyzed by SigmaStat software (Jandel Scientific, San
Rafael, CA). The significance of differences was tested where
appropriate by Student's t test or analysis of variance,
and p < 0.05 was taken to be significant.
 |
RESULTS |
Development of System to Overexpress MCAT-1 Receptors in Human
Cells
MCAT-1 Transgene Expression--
A total of 14 HuH-7.MCAT-1 cell
clones were analyzed 4 weeks after transfection with the plasmid
pcDNA3MCAT-1. In all positive clones, steady-state
L-[3H]arginine accumulation was markedly
increased compared with the untransfected parental cells (Table
I). From among these cell clones, the
HuH-7.MCAT-1.7 clone was randomly chosen for detailed studies because
these cells expressed the introduced MCAT-1 cDNA with increased
susceptibility to ecotropic retroviral infection, although not uniquely
so. DNA transblot analysis showed that the HuH-7.MCAT-1.7 cells
contained integrated MCAT-1 cDNA sequences that were absent in the
parental HuH-7 cells (Fig.
1A). Although additional faint
hybridization bands could have represented cross-hybridization with
partially homologous endogenous sequences, these were not further
characterized. RNA transblots showed that HuH-7.MCAT-1.7 cells
contained mRNA transcripts that hybridized with a full-length MCAT-1 cDNA probe and corresponded to the 2.4-kilobase pair size of
transfected MCAT-1 cDNA (Fig. 1B). In addition,
10
5 M dexamethasone up-regulated the
steady-state abundance of this transgene transcript by severalfold. The
RNase protection assay showed that HuH-7.MCAT-1.7 cells expressed
unique mRNA transcripts hybridizing with the MCAT-1 riboprobe that
were absent in parental HuH-7 cells (Fig. 1, C and
D). In addition, the steady-state abundance of the MCAT-1
transgene transcript was up-regulated by severalfold after exposure to
10
5 M dexamethasone. Interestingly, RNase
protection showed that
-actin mRNAs were also up-regulated by
dexamethasone in HuH-7.MCAT-1.7 cells, although the significance of
this observation was unclear. Direct demonstration of the MCAT-1 gene
product in the HuH-7.MCAT-1.7 cells was impossible due to the
nonavailability of a reliable antibody for transblotting or
immunoprecipitation, despite the use of an antibody for immunostaining
(26).
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Table I
L-Arginine accumulation in HuH-7.MCAT-1 cell clones
All experiments were carried out in triplicate at least; p
values were calculated with Student's t test.
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Fig. 1.
Analysis of MCAT-1 transgene. A,
Southern blot analysis of MCAT-1 cDNA samples and genomic DNA from
HuH-7.MCAT-1.7 cells. Lane 1, 1 pg of
32P-labeled MCAT-1 cDNA probe; lane 2, 0.1 pg of 32P-labeled MCAT-1 cDNA probe; lane 3,
DNA from HuH-7.MCAT-1.7 cells; lane 4, DNA from parental
HuH-7 cells. The genomic DNAs were digested with BamHI and
EcoRI, which would release the MCAT-1 transgene with the
expected size of ~2.4 kilobase pairs (Kb). B,
RNA transblot analysis showing MCAT-1 mRNA expression. Panel
A shows expression of the 2.4-kilobase pair transgene in
HuH-7.MCAT-1.7 cells (lane 1), but not in parental HuH-7
cells (lane 2). Upon exposure to 10 5
M dexamethasone (panel B), transgene expression
increased (lane 1) compared with the base-line expression in
untreated (lane 2) HuH-7.MCAT-1.7 cells. C,
polyacrylamide gels after RNase protection showed -actin mRNAs
(250 base pairs), but no MCAT-1 mRNAs (lane 3) in
parental HuH-7 cells. HuH-7.MCAT-1.7 cells additionally contained
MCAT-1 mRNA (expected size of 720 base pairs) (lane 1).
In addition, after stimulation with 10 5 M
dexamethasone for 48 h, the abundance of both MCAT-1 and -actin
mRNAs increased in HuH-7.MCAT-1.7 cells (lane 2).
D, slot-blot analysis showing the relative abundance of the
mRNAs after RNase protection in HuH-7.MCAT-1.7 cells (rows 1 and 2) and parental HuH-7 cells (row 3).
Row 4 contained the probes used for hybridization with RNA
samples. The bands in the left lane indicate
-actin mRNAs, and those in the right lane indicate
MCAT-1 mRNAs in the same RNA samples. The HuH-7.MCAT-1.7 cells
showed MCAT-1 mRNA expression under base-line culture conditions
(row 1), with severalfold increased mRNA accumulation
after dexa-methasone treatment (row 2). In contrast, no
MCAT-1 transcripts were present in HuH-7 cells (row 3). The
data were reproducible in three independent experiments.
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Effect of MCAT-1 Activity on Amino Acid Transport and
Uptake--
The transport and intracellular retention of arginine were
markedly increased in HuH-7.MCAT-1.7 cells. The initial velocity of
L-arginine uptake appeared to be greater in HuH-7.MCAT-1.7 and NIH 3T3 cells compared with HuH-7 cells (Fig.
2A). The relevant parameters
of the transport kinetics obtained from three independent experiments
are summarized in Table II. The
L-arginine uptake showed saturable kinetics in parental
HuH-7 cells, with Vmax = 3.0 ± 0.5 nmol/mg
of protein/min and Km = 257 ± 16 µM. In contrast, the Vmax of
L-arginine transport was significantly greater in
HuH-7.MCAT-1.7 cells (mean, 160% greater), indicating higher capacity
as well as higher affinity for arginine transport. In contrast, in the
NIH 3T3 cells, which exhibit remarkable avidity for ecotropic
retroviruses, the L-arginine uptake was characterized by
even greater affinity, but with a lower capacity (mean, 77% less), in
comparison with the parental HuH-7 cells. Interestingly, when compared
with NIH 3T3 cells, the Vmax and
Km of arginine transport in the HuH-7.MCAT-1.7 cells
were significantly different (p < 0.03 and
p < 0.002, respectively), with the HuH-7.MCAT-1.7 cells showing lower affinity and higher apparent capacity for L-arginine transport. The L-lysine transport
analysis demonstrated a greater initial velocity of
L-lysine uptake in HuH-7.MCAT-1.7 cells (Fig.
2B). The mean Vmax (7.03 ± 0.52 nmol/mg of protein/min) for L-lysine in these cells was
significantly greater (161%) than that in parental HuH-7 cells
(Vmax = 4.36 ± 0.21 nmol/mg of
protein/min; p < 0.01), indicating again a higher
affinity and capacity for another cationic amino acid. We found that
arginine uptake could not be saturated in primary rat hepatocytes,
confirming previous studies (12) and further contrasting with the
arginine transport observed in HuH-7.MCAT-1.7 cells. On the other hand,
the proline transport was similar in HuH-7 and HuH-7.MCAT-1.7 cells,
with Vmax = 2.35 ± 0.11 versus
2.38 ± 0.14 nmol/mg of protein/min and Km = 0.138 ± 0.10 versus 0.124 ± 0.09 mM,
respectively (p = not significant) (Fig.
2C).

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Fig. 2.
Amino acid uptake in cells. A,
shown are the representative kinetics of
L-[3H]arginine uptake in HuH-7,
HuH-7.MCAT-1.7, and NIH 3T3 cells. The initial velocity of arginine
uptake appeared to be greater in the latter two cell lines compared
with HuH-7 cells. The Vmax and apparent
Km were significantly different in the cells tested
(see Table II and "Results" for discussion). Six independent
experiments produced similar results. B, the kinetics of
L-[3H]lysine uptake in HuH-7.MCAT-1.7 cells
were also significantly different from those in HuH-7 cells and
resembled the L-arginine transport parameters.
C, in contrast with L-arginine or
L-lysine uptake, no differences were apparent in
L-[14C]proline uptake in HuH-7 and
HuH-7.MCAT-1.7 cells.
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MCAT-1 overexpression is known to increase the steady-state
intracellular accumulation of L-arginine as shown in
Xenopus oocytes and mink fibroblasts (27), although the
previous systems were dissimilar to ours. Utilizing retention of
L-[3H]arginine allowed us to conveniently
examine the hormonal regulation of MCAT-1 activity. Our studies of
steady-state arginine accumulation showed that L-arginine
retention in HuH-7.MCAT-1.7 cells was markedly increased compared with
HuH-7 cells (>300%, p < 0.001) (Fig.
3). The increase in
L-[3H]arginine accumulation was competitively
inhibited by unlabeled arginine. Although increased amino acid
transport activity was clearly associated with greater intracellular
L-arginine retention, precise mechanisms underlying this
process, such as the role of specific cell compartments, were undefined
and will require further study.

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Fig. 3.
Steady-state L-arginine uptake in
cells. When cells were exposed to the
L-[3H]arginine tracer in the presence of 0.02 mM arginine in culture medium, there was a significantly
greater L-[3H]arginine accumulation in
HuH-7.MCAT-1.7 cells and NIH 3T3 cells compared with HuH-7 cells. In
the presence of greater concentrations of unlabeled arginine in the
culture medium, however, uptake and steady-state
L-[3H]arginine accumulation were
competitively inhibited in all three cell lines.
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When HuH-7.MCAT-1.7 cells were cultured for 24 h in
arginine-deficient RPMI 1640 medium containing 10
6 or
10
5 M dexamethasone, steady-state
accumulation of L-arginine significantly increased by up to
~2-fold (Table III). Interestingly,
10
4 M norepinephrine also increased
L-arginine accumulation by ~1.4-fold in HuH-7.MCAT-1.7
cells, whereas insulin alone did not change steady-state arginine
accumulation. Finally, in response to dexamethasone and HGF, the
steady-state accumulation of L-arginine was maximally increased by up to ~3-fold. These results indicated that the MCAT-1 activity in HuH-7.MCAT-1.7 cells could be modulated by hormonal stimulation because the CMV promoter used for driving transgene expression is up-regulated by dexamethasone (28).
MCAT-1 Expression Does Not Alter Cell Proliferation
States--
The doubling times of HuH-7.MCAT-1.7 cells were similar to
those of HuH-7 cells, 28 ± 2 versus 30 ± 3 h, respectively (p = not significant). In contrast, the
doubling times of both these cell lines significantly differed from
those of NIH 3T3 fibroblasts, which doubled in 18 ± 3 h
(p < 0.001). When [3H]thymidine
incorporation was measured as a marker of DNA synthesis at 24 and
72 h after plating 2 × 104 HuH-7 or
HuH-7.MCAT-1.7 cells, again no significant differences were observed
(Fig. 4). Finally, flow cytometry of
propidium iodide-stained HuH-7 and HuH-7.MCAT-1.7 cells showed similar
cell cycle profiles, confirming that MCAT-1 overexpression did not
alter the proliferative status of the cell clone (data not shown).

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Fig. 4.
Proliferation capacity of HuH-7 and
HuH-7.MCAT-1.7 cells. Serial cell counts (shown by the
lines) showed that the two cells had similar doubling times
(HuH-7 cells, 30 ± 3 h; and HuH-7.MCAT-1.7 cells, 28 ± 2 h) (p = not significant). In addition, there was
no difference in DNA synthesis rates during log phase growth, with
similar incorporation of [3H]thymidine into DNA.
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Overexpression of the Heterologous Retroviral Receptor and Hepatic
Gene Transfer
The transduction rates were dependent upon virus concentration.
The ecotropic LacZ retrovirus transduced 21 ± 3, 93 ± 2, and 98 ± 1% NIH 3T3 cells at m.o.i. values of 0.2, 2, and 25, respectively. In contrast, HuH-7.MCAT-1.7 cells showed gene expression
in 5 ± 1, 18 ± 2, and 65 ± 2% cells at corresponding
m.o.i. values of 0.2, 2, and 25, respectively (p < 0.02; analysis of variance). The number of blue-stained cells in
parental HuH-7 cells was essentially unaffected, with at most 1.0 ± 0.7% cells at the highest m.o.i. of 25. Further testing with single
HuH-7.MCAT-1.7 cell clones (n = 25) derived by
dilutional cloning showed susceptibility to the ecotropic retrovirus
vector in all cell clones, with 7-35% cells staining blue at a m.o.i.
of 2. In contrast, the parental HuH-7 cells showed LacZ expression with
a m.o.i. of 2 in only 0.7 ± 0.8% cells and, despite the maximal
m.o.i. of the virus used, in <1.5% cells. In contrast with these
results, infection with the amphotropic LacZ virus was similar in both
HuH-7 and HuH-7.MCAT-1.7 cells (55 ± 8 versus 57 ± 7% blue-stained cells, respectively; p = not
significant).
Up-regulation of Viral Receptor Expression and Gene
Transfer--
When HuH-7.MCAT-1.7 cells were infected along with
hormone treatments, gene transfer improved significantly (Fig.
5). Exposure of cells to either
10
5 M dexamethasone alone or with 20 ng/ml
HGF increased ecotropic retroviral transfer in HuH-7.MCAT-1.7 cells by
up to 2- and 4-fold, respectively (p < 0.001). In
contrast, exposure of HuH-7.MCAT-1.7 cells to the ecotropic virus in
the presence of insulin alone, norepinephrine alone, or dexamethasone
at concentrations <10
6 M did not have any
effect upon gene transfer efficiency. Exposure of either HuH-7 cells or
NIH 3T3 cells to the hormones had no effect upon retroviral gene
transfer. On the other hand, NIH 3T3 cells became unhealthy and started
to detach from tissue culture dishes in the presence of dexamethasone.
The differential response of fibroblast- and hepatocyte-derived cells
to hormonal stimulation was not surprising because unlike hepatocytes,
dexamethasone might have had a negative effect upon gene expression or
altered differentiation states in fibroblasts as suggested by previous
studies (29, 30). Assays of [3H]thymidine incorporation
into DNA indicated that after exposure to 20 ng/ml HGF for 24 h,
DNA synthesis in HuH-7 and HuH-7.MCAT-1.7 cells increased by ~2-fold
(p < 0.05).

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Fig. 5.
Susceptibility of various cells to
ecotropic retroviral gene transfer. The data show representative
examples of gene transfer in parental HuH-7 cells (A), NIH
3T3 cells (B), HuH-7.MCAT-1.7 cells (C), and
HuH-7.MCAT-1.7 cells treated with 10 5 M
dexamethasone and 20 ng/ml HGF for 24 h (D). All cells
were incubated with the ecotropic LacZ retrovirus (m.o.i. = 2) for
5 h; fresh medium was switched; and cells were stained for LacZ
activity 72 h later. The efficiency of retroviral infection was
maximal in NIH 3T3 cells. However, in contrast with HuH-7 cells, which
were essentially negative, LacZ expression was significantly greater in
HuH-7.MCAT-1.7 cells and further increased with hormonal up-regulation
of MCAT-1 activity.
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Abolition of Cell Cycling Interferes with Retroviral Gene
Transfer--
The AdMad virus allowed us to dissect the roles of
receptor overexpression and cell proliferation in retroviral gene
transfer. When HuH-7 cells were incubated with AdMad, there was
increased expression of the Mad protein as demonstrated by
immunoblotting (data not shown). Studies with the Ad
gal virus showed
that virtually 100% of the HuH-7 or HuH-7.MCAT-1.7 cells could be
infected by the adenovirus. Upon exposure of log phase growth HuH-7
cells to the AdMad virus, the cell number increased only by 28 ± 6% compared with 242 ± 47% in untreated control HuH-7 cells
(p < 0.001). Flow cytometry corroborated these
findings and demonstrated accumulation of AdMad-treated cells primarily
in G0/G1 (Fig.
6). However, despite exposure of the
HuH-7.MCAT-1.7 cells to AdMad, the MCAT-1 activity was unchanged, as
shown by accumulation of [3H]arginine in response to
stimulation by 10
5 M dexamethasone (6312 ± 430 versus 6188 ± 168 dpm/µg of protein/2 h;
p = not significant). Finally, when asynchronously
cycling HuH-7.MCAT-1.7 cells were first infected with the AdMad virus and 72 h later infected with the ecotropic LacZ virus, gene
transfer was virtually abolished (32 ± 3 versus 1 ± 0.6% blue-stained cells in control and AdMad-treated cells,
respectively; p < 0.001). In contrast, infection of
the HuH-7.MCAT-1.7 cells with the adenovirus containing antisense
hepatitis B virus sequences had no effect upon ecotropic retroviral
infection. Similarly, infection with an amphotropic LacZ retrovirus of
HuH-7.MCAT-1.7 cells previously exposed to AdMad resulted in no gene
transfer.

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Fig. 6.
Flow cytometric demonstration of cell cycling
after exposure to AdMad. Analysis of DNA content in cells
indicated that S phase cells were more abundant in control
HuH-7.MCAT-1.7 cells not exposed to the AdMad virus (A),
whereas HuH-7.MCAT-1.7 cells exposed to the AdMad virus were enriched
in G0/G1 (B). This change in cell
cycling did not affect MCAT-1 activity in HuH-7.MCAT-1.7 cells, however
(see "Results") (C).
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DISCUSSION |
Our findings are in agreement with retroviral receptor
overexpression alone without DNA synthesis being insufficient for
improving retroviral gene transfer. The studies showed that retroviral
gene transfer was virtually abolished when either of these processes was abrogated. We demonstrated that resistance of human cells to
ecotropic retroviruses was overcome by MCAT-1 overexpression, which is
in agreement with results obtained in mink fibroblast and Chinese
hamster ovary cells that are also resistant to ecotropic retroviruses
in their native states (4).
Although our studies were not directed at detailed analysis of the
cationic amino acid transport mechanism, we nonetheless found that
MCAT-1 overexpression increased L-arginine and
L-lysine transport in a high affinity fashion in
HuH-7.MCAT-1.7 cells. In contrast, the parental HuH-7 cells exhibited a
lower affinity for L-arginine and L-lysine
transport, which probably indicates the presence of alternative
transporters. Additional MCAT transporters, designated MCAT-2 and
MCAT-2A, have been identified (31, 32), but no viruses utilizing these
transporters as their cellular receptors have yet been recognized. The
MCAT-2A gene product, which shares substrate specificity with MCAT-1,
but exhibits much higher capacity for cationic amino acids in
hepatocytes (~10-fold greater) (32), could be one such candidate. In
fact, the total L-arginine transport in our HuH-7.MCAT-1.7
cells most likely represents the sum effect of MCAT-1 plus other
transporters. Therefore, by using the L-arginine transport
alone, MCAT-1 activity cannot possibly be directly compared between the
HuH-7.MCAT-1.7 and NIH 3T3 cells. However, increased
L-arginine transport did serve as a useful surrogate
reporter for demonstrating MCAT-1 activity in our cells, although the
efficiency of ecotropic retroviral infection was the best measure of
receptor activity. Hormonal treatment increased retroviral infection in
HuH-7.MCAT-1.7 cells, which was most likely due to up-regulated
transgene expression since the CMV promoter is regulated by
dexamethasone, although hormones may also up-regulate endogenous
receptor activity (11, 29). Judging from L-arginine transport, however, it would appear that the endogenous MCAT-1 receptor
in NIH 3T3 cells was far more efficient in its retroviral receptor
function than the MCAT-1 in HuH-7.MCAT-1.7 cells. Whether this was
directly related to quantitative differences in MCAT-1 expression or
qualitative differences, e.g. the presence or absence of
unidentified regulatory subunits that improve receptor binding to
specific viral domains, is unknown. Overexpression of other retroviral
receptors showing low base-line organ expression, e.g. the
amphotropic gibbon ape leukemia virus receptor, which shares a unique
membrane-spanning domain determining retroviral infection with MCAT-1,
can render murine cells susceptible to the appropriate virus (34-36).
Analysis of the Moloney murine leukemia virus indicates that 246 amino-terminal gp70 residues contain the MCAT-1-binding domain and that
the variable region designated VRA regulates direct interactions with
the receptor and thus the host range (37-39), whereas resistance of
Chinese hamster ovary cells to ecotropic Moloney murine leukemia virus
is due to glycosylation of the hamster chloramphenicol
acetyltransferase, which may transiently be overcome by the
N-glycosylation inhibitor tunicamycin (40). On the other hand, in cells infected by retroviruses, the turnover of MCAT-1 itself
is unchanged (41), and our results indicate that ecotropic retroviruses
require specific receptor domains that may not be substituted by
alternative cellular cationic amino acid transporters, which is
consistent with the resistance of both HepG2 and HuH-7 cells to
infection with ecotropic retroviruses.
It is remarkable, from a teleological point of view, that for entry
into cells, retroviruses exploit an ubiquitous transporter, which is
up-regulated during cell proliferation. This two-headed approach is
most efficient because of the obvious advantages for viral entry and
proviral integrations. Although rapidly cycling cells, such as
progenitor or stem cells, could potentially be infected with retroviral
vectors (42), this is not the case with adult hepatocytes, which are
proliferatively quiescent and restricted mostly to
G0/G1, thus limiting retroviral receptor expression as well as proviral integrations. In view of the
attractiveness of liver-directed gene therapy for metabolic disorders,
strategies to improve hepatic gene transfer include ways to increase
retroviral titer, which is currently limited to 1 × 106 to 107 virion particles/ml. However, the
dose-dependent increases in retroviral gene transfer in
cells overexpressing viral receptors in our studies support such a
strategy and need for further work in this area. We believe that
additional strategies based upon overexpression of viral receptors
alone will also be successful, as shown by increased retroviral gene
transfer upon hormonal up-regulation of MCAT-1 activity in our studies.
Clearly, however, retroviral gene transfer will be most efficient when
viral receptors are overexpressed in the setting of ongoing cell
proliferation. While overexpression of retroviral receptors in the
presence of increasing viral titer but constant proliferative activity
was effective, abolition of cell cycling by exposure of cells to the
Mad transcription factor prevented proviral integrations and gene
transfer.
The translational implications of these findings are that if retroviral
receptors were iatrogenically overexpressed in cells capable of high
grade proliferative activity, such as stem cells, gene transfer would
be very efficient, with permanent gene expression in daughter cells. In
addition, overexpression of heterologous viral receptors in tissues,
such as the liver, could offer one way to target retroviral vectors in
a tissue-specific manner. In view of their broad activities,
amphotropic or xenotropic retroviral receptors will be less suited for
such a task in humans, whereas the MCAT-1 receptor might well be a
candidate (43). This could potentially be accomplished with a dual
vector strategy, e.g. by first transiently expressing the
MCAT-1 retroviral receptor with efficient adenoviral, herpes simplex
virus-1, or other vectors, followed by exposure to the retrovirus
containing a therapeutic gene. The potential of dual vector approaches
for hepatic gene transfer has begun to be addressed (15). However, the
strategy to overexpress retroviral receptors by itself will be
ineffective in proliferatively quiescent hepatocytes, which will also
require a mitogenic stimulus for inducing cell proliferation and
proviral integrations. Nonetheless, the first arm of the strategy
should be quite successful because hepatocytes contain abundant
receptors for transduction with adenoviral or herpes simplex virus-1
vectors, and the second arm would be facilitated by recent insights
into liver growth control by exogenously administered growth factors, such as HGF, transforming growth factor-
, epidermal growth factor, and others (5, 44, 45). Use of an ecotropic retrovirus for gene
transfer via such a dual vector strategy should be specially safe for
laboratory personnel and care givers. However, the recipient could
potentially be susceptible to murine retroviruses, although such an
exposure would be temporary if the introduced MCAT-1 cDNA were to
be localized episomally and survive transiently, as would be expected
with adenoviral and physical gene vectors.
We thank Dr. J. M. Cunningham (Howard
Hughes Medical Institute) for providing the pJET plasmid containing the
MCAT-1 cDNA, Drs. R. A. DePinho (Albert Einstein College of
Medicine) and P. Nisen (Southwestern Medical Center, University of
Texas) for providing the recombinant AdMad virus stock, and Genentech
Corp. (South San Francisco, CA) for supplying purified recombinant
human HGF.