From the Division of Medical Oncology, Brown University School of Medicine and Graduate Program in Pathobiology, Rhode Island Hospital, Providence, Rhode Island 02903
Received for publication, September 8, 2000, and in revised form, February 1, 2001
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ABSTRACT |
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L-amino acid transporter-1
(LAT1) is a highly conserved gene identified as a light chain of the
CD98 amino acid transporter and cellular activation marker. In our
previous studies we found increased expression of LAT1 in primary human
cancers. We have demonstrated also that LAT1 response to arginine
availability is lost in transformed and tumorigenic cells such that
expression is constitutively high. System L-amino acid
transport activity correlates with changes in LAT1. To assess the
functional relevance of increased LAT1 expression and the requirement
for 4F2 heavy chain, we overexpressed these CD98 subunits together and
separately in nontransformed hepatocytes and fibroblasts. Antigen tags
in the expression constructs confirmed that expressed proteins were localized to both cytoplasmic and plasma membrane locations within the
cells. Overexpression of LAT1 alone in mouse hepatocytes, but not
fibroblasts, was sufficient to increase system L transport, and these cells displayed a growth advantage in conditions of limited
arginine. Our results suggest that loss of regulation leading to
constitutive expression of LAT1 can contribute to oncogenesis. We
hypothesize that the altered LAT1 expression observed in
hepatocarcinogenesis gives cells a growth or survival advantage through
increased transport activity in a tumor microenvironment of limited
amino acid availability.
LAT1/TA1 and its homologs have been identified by multiple
investigators as one of several alternate light chains of the CD98 amino acid transporter and cellular activation marker (1-6). TA1 was
cloned in our laboratory on the basis of its differential expression
between rat hepatoma cells and normal adult rat liver and is identical
to the C terminus (amino acids 272-512) of LAT1 (7). The CD98 complex
consists of an 80-kDa heavy chain (4F2hc) and a 40-45-kDa light chain
(8). CD98 has been implicated in a variety of functions including amino
acid transport, cell survival, integrin activation, and cell fusion
(9-12). Various light chains have the potential to form a complex with
heavy chain depending on the cell type and intracellular localization.
Additional light chains have been identified and designated
y+LAT-1 (13-15), y+LAT-2 (13, 16), LAT-2 (17,
18), xCT (19), 4F2-lc6 (20), and ASC-1 (21). The CD98 complex can
mediate various amino acid transport systems in Xenopus
oocytes depending on which CD98 light chain (CD98lc) is associated with
the CD98 heavy chain (CD98hc) (3-6, 13). Co-injection of cRNA for
TA1/LAT-1 and 4F2hc has been shown to mediate system L
transport of large neutral amino acids with branched or aromatic side
chains in Xenopus oocytes (3, 5).
We have found increased levels of LAT1 mRNA in both rat and human
carcinomas and in the carbon tetrachloride model of liver injury/regeneration (7, 22-24). Adaptive regulation of message for
LAT1 but not 4F2hc was observed in response to arginine levels in
normal hepatic cells (25). Loss of LAT1 regulation with corresponding increased system L transport was associated with malignant
progression, suggesting that up-regulation of LAT1 or functional CD98
complex contributes to the neoplastic phenotype. Hara et al.
(26) have shown that NIH3T3 cells transfected to overexpress 4F2hc
demonstrated classic properties of malignant cells including higher
saturation density, growth in soft agar, and tumor formation in athymic
mice. Researchers from the same laboratory demonstrated more recently that transformation of BALB3T3 cells by overexpression of 4F2hc requires its association with an unidentified light chain (27).
We are interested in the regulation and role of LAT1 and CD98-related
molecules in transformation and carcinogenesis. To examine the
relationship of cell type to overexpression of these molecules and
associated transport properties, we transiently overexpressed LAT1 and
4F2hc alone or together in nontransformed mouse hepatocyte and
fibroblast cell lines. We found that overexpression of LAT1 alone was
sufficient to increase system L transport activity
significantly in the epithelial cells but not in the fibroblasts. In
contrast, increases in transport activity in fibroblasts required
coexpression of both LAT1 and 4F2hc. Hepatic cells overexpressing LAT1
displayed a growth advantage relative to control cells under conditions of limited arginine. We postulate that differences in LAT1 expression and response to amino acid availability influence cell growth and
survival properties and that the LAT1-CD98 pathway may represent a
unique therapeutic target for cancer intervention.
Cell Culture--
AML12 cells, a normal mouse hepatocyte line,
were provided kindly by Dr. Nelson Fausto (Department of Pathology,
University of Washington) and maintained in Dulbecco's modified
Eagle's medium/Ham's F-12 medium (Life Technologies, Inc.) containing
10% fetal bovine serum and supplemented with insulin,
transferrin, and selenium (ITS+) at 1 ml/liter medium
(Becton Dickinson) and 0.1 µM dexamethasone. NIH3T3 cells
were cultured routinely in Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum. A custom formulation of Chee's
essential medium (CEM)1 that
was demonstrated to maintain long term differentiated cell function
in vitro was provided kindly by Dr. Hugo Jauregui
(Department of Pathology, Rhode Island Hospital) and prepared as
described previously (28). Arginine was not present in this custom
formulation and was added from stock solutions. For experiments in
which gene expression was assayed as a function of arginine
availability, cells were seeded into T-75 flasks with CEM containing
5% fetal bovine serum with and without arginine (25). Previous
experiments have shown no difference in results with dialyzed or
nondialyzed serum (25). Medium was changed every day. Viability was at
least 90% in all cases.
Functional Expression of cDNAs in AML12 and NIH3T3
Cells--
Rat LAT1 cDNA was subcloned into the pFLAG-5a
expression vector (Sigma) and sequenced to confirm the correct sequence
and the addition of the FLAG epitope tag. pL(CD98-HA)IZ expression construct and monoclonal antibody HA.11 (Babco) were provided kindly by
Dr. Tatiana Kolesnikova and Dr. Martin Hemler (Dana Farber Cancer
Institute, Harvard Medical School). pL(CD98-HA)IZ expresses human
4F2hc/CD98 heavy chain with a hemagglutinin epitope tag. pEGFP-F
(CLONTECH) encodes farnesylated enhanced
green fluorescent protein.
For transport assays, AML12 cells and NIH3T3 cells were seeded,
respectively, at 2 × 105 and 7.5 × 104 cells/well of a 24-well plate 24 h prior to
transfection. For immunofluorescence, AML12 and NIH3T3 cells were
seeded, respectively, at 4 × 105 and 1.5 × 105 cells/chamber of a slide chamber 24 h prior to
transfection. Transfection was carried out with LipofectAMINE Plus
reagent (Life Technologies, Inc.). For a 24-well plate, 0.6 µg of
DNA, 3 µl of Plus reagent, and 1.5 µl of LipofectAMINE were used
per well. For a chamber slide, 1.2 µg of DNA, 6 µl of Plus reagent,
and 3 µl of LipofectAMINE were used per chamber. The functional
expression of each cDNA was analyzed 48 h post-transfection by
measuring radiolabeled amino acid uptake and by immunofluorescence in
AML12 and NIH3T3 cells. Cotransfection with pEGFP-F was used for every experiment to monitor transfection efficiency by counting fluorescent cells expressing GFP. Transfection efficiency routinely approached 40 and 50% for NIH3T3 and AML12, respectively.
Immunofluorescence--
Expression of exogenous LAT1 and 4F2 was
monitored by indirect immunofluorescence microscopy using antibodies
directed to the epitope tag present on each protein. M2 antibody
(Sigma) was used to detect FLAG-tagged LAT1, whereas HA.11 antibody
(Babco) was used to detect hemagglutinin-tagged 4F2hc. Briefly, AML12 and NIH3T3 cells were transfected with pFLAG-LAT1 or pL(CD98-HA)IZ, and
at 48 h after transfection, slides were fixed in cold acetone. Slides were blocked with 1% normal goat serum in PBS and then incubated with either M2 (mouse anti-FLAG) antibody at 10 µg/ml or
HA.11 (mouse anti-hemagglutinin) antibody at 1:100 dilution in 1%
normal goat serum in PBS. Slides were washed with PBS, blocked again,
and incubated with goat anti-mouse secondary antibody (Sigma) at 1:100
dilution in 1% normal goat serum in PBS. After a subsequent wash and
coverslipping, slides were examined on a Nikon Microphot-EPI-FL fluorescence microscope.
Expression of endogenous LAT1 was detected using affinity-purified
anti-peptide antibody to the NH2 terminus of LAT1 at
12 µg/ml. MacVector sequence analysis of the rat LAT1 coding region was used to select a region with little or no homology to other CD98
light chains and with high conservation to human LAT-1 and high
hydrophilicity. Peptide 20-30, EERQAREKML, near the NH2
terminus scored highest for these qualities and was synthesized
commercially with an NH2-terminal cysteine for conjugation
to keyhole lympet hemocyanin and used to immunize two rabbits following
a conventional immunization schedule (Sigma-Genosys). The specificity
of this antibody was demonstrated by absorption with the immunizing
peptide (data not shown). Expression of endogenous 4F2hc was detected using an anti-4F2hc rabbit anti-peptide antibody (1:100) kindly provided by Dr. Suresh S. Tate (Cornell University Medical College, NY). This polyclonal antiserum was raised against a 92-amino acid peptide fragment (Lys-114-Tyr-205) of the rat glioma cell 4F2hc (29).
Normal rabbit serum at the same concentration or dilution was used as a
negative control (data not shown). Normal liver and GP7TB tumor cells
were used as negative and positive controls, respectively, for LAT1
antibody reactivity (data not shown).
RNA Preparation and Northern Blot Analysis--
Total RNA was
isolated using the guanidinium isothiocyanate/cesium chloride method
(30) for rat placenta and a modification of that method for cultured
cell lines (Totally RNA KitTM, Ambion, Austin, TX). RNA was isolated
from cultured cells after subculture in CEM for the relevant time
points. Aliquots (12 µg) of total RNA were size-fractionated on 1%
agarose/formaldehyde gels as described previously (22). After
electrophoresis, gels were equilibrated in 1 M ammonium
acetate, and RNA was transferred to Nytran nylon membranes (Schleicher
and Schüll). Blots were baked for 2 h at 80 °C and
hybridized at 65 °C according to Church and Gilbert (31). Rat
TA1 p900, a fragment corresponding to nucleotides 816-1536
of the full-length rat LAT-1 inserted into Bluescript SK vector, was
labeled with [32P]dCTP (3000 Ci/mmol, PerkinElmer Life
Sciences) by random primed labeling (Roche Molecular Biochemicals) for
use as a probe. Blots wrapped in plastic wrap were exposed to x-ray
film (Eastman Kodak Co.) at Amino Acid Transport Assays--
The transport of radiolabeled
amino acids by cell monolayers was performed using a modification of
the cluster-tray method developed by Gazzola et al. (33) and
described by Kilberg (34). All 3H-labeled amino acids were
purchased from PerkinElmer Life Sciences and unlabeled amino acids were
purchased from Sigma. Cells were near confluent for the transport
assay. Transport assays were performed as described previously (25).
Briefly, before the transport assays, cells were rinsed with warm
Na+-free KRP buffer, in which the sodium-containing salts
were iso-osmotically replaced with choline, to remove extracellular
Na+ and amino acids. Cells were incubated in warm
Na+-free KRP buffer for 10 min to deplete intracellular
amino acids. The uptake of radiolabeled amino acids (5 µCi of
[3H]amino acid/ml) at 50 µmol/liter in either 200 µl
of Na+-free KRP buffer or sodium KRP was measured for
30 s at 37 °C. Preliminary experiments indicated that uptake of
each 3H-labeled amino acid was linearly dependent on
incubation time up to at least 3 min; therefore, uptake was measured
for 30 s (data not shown). In inhibition experiments, excess cold
2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) was added to the
uptake buffer at 10 mM final concentration. Uptake was
terminated by washing the cells rapidly four times with 1 ml/well of
ice-cold Na+-free KRP buffer. After the trays were allowed
to dry, the cells were incubated for 1 h with 0.2 ml/well of 0.2%
(w/v) SDS plus 0.2 N NaOH to release intracellular radioactivity. A
0.1-ml aliquot from each well was neutralized with 0.1 ml of 0.2 N HCl and quantified in a Beckman LS 6000SC liquid
scintillation counter. The remaining 0.1 ml was analyzed for protein
content using the BCA protein assay reagent (Pierce). Transport
velocities were calculated from radioactive counts, specific activities
of uptake mixes, and protein absorbance values and were expressed as
picomoles amino acid transported per mg of protein per min
(averages ± S.E. of Proliferation Assay and Cell Cycle Analysis--
Cells were
seeded into 6-well plates, transfected with plasmid DNA, and then
cultured in CEM containing 5% fetal bovine serum without arginine. The
medium was changed every day. Transfection efficiency was monitored by
GFP expression and immunofluorescence. Cells were counted by the trypan
dye exclusion test at the beginning of the experiment as well as 24 and
48 h after culture in CEM without arginine. Cell number was
expressed as averages ± S.E. of three or more separate
determinations. The experiment was repeated with independent
transfections to show qualitatively the same results. An aliquot of
cells from each condition was saved for cell cycle analysis. These
cells were stained with 1× propidium iodide stain (50 µg/ml
propidium iodide, 0.1% Nonidet P-40, and 0.1% sodium citrate in
H2O) for ~10 min. Cell cycle analysis was performed using
a Becton Dickinson FACSORT flow cytometer, and data were analyzed with
Cell Quest software.
Transfection of AML12 and NIH3T3 Cells--
AML12 cells, an
immortalized, nontransformed, nontumorigenic, well differentiated
hepatocyte cell line, was established from the liver of transgenic mice
overexpressing transforming growth factor- Immunofluorescence Detection of Exogenous and Endogenous LAT1 and
4F2hc in NIH3T3 and AML Cells--
NIH3T3 and AML12 cells were
transfected with either pFLAG-LAT1 or pL(CD98-HA)IZ to verify
expression of exogenous protein by immunofluorescence in addition to
transport activity (Fig. 2, A
and B). Both FLAG-tagged LAT1 and
hemagglutinin-tagged 4F2hc were detected in transfected NIH3T3 and
AML12 cells. The staining pattern indicated significant colocalization
in both cell types. Staining was observed at the cell surface as well
as within the cytoplasm. Anti-peptide antibodies were used to detect
endogenous 4F2hc and LAT1 staining in both AML12 and NIH3T3 cells (Fig.
2, C and D). The LAT1 epitope is on the
NH2 terminus of the molecule, which is predicted to be
cytoplasmic (38). The antibody to 4F2hc is directed to a predicted
ectodomain (38). Intense staining was observed for 4F2hc in AML12 and
NIH3T3 cells and for LAT1 in NIH3T3 cells. Staining for LAT1 was very
weak in AML12 cells. The staining pattern for endogenous 4F2hc and LAT1
was very similar to staining for exogenous 4F2hc and LAT1 with both
cytoplasmic and cell membrane staining. We observed similar
immunofluorescent staining for all transfections.
Overexpression of LAT1 Is Sufficient to Increase System
L Transport Activity in AML12 Cells--
In previous
studies using a panel of hepatic cell lines with specific differences
in transformation and tumorigenicity, we found that LAT1 but not 4F2hc
correlated with transformation, tumorigenicity, and increases in system
L transport activity (25). To determine the functional
relevance of overexpression of LAT1 and requirement for 4F2hc in
nontransformed hepatocytes, we overexpressed LAT1 and/or 4F2hc in AML12
cells and measured transport activity. Transport activity for systems
L and y+ were found to be sodium-independent
(data not shown). As shown in Fig.
3a, overexpression of LAT1
increased system L-specific transport activity 2-fold
(significant at p < 0.005) relative to 4F2hc or empty
vector transfection. Overexpression of LAT1 and 4F2hc did not increase
transport activity above that of LAT1 alone. As expected, addition of
excess cold BCH inhibited leucine transport, consistent with system
L activity. Arginine transport (system y+) was
not significantly different in any of the transfection conditions (Fig.
3b) and served as a control for any nonspecific influence of
the transfection conditions.
Overexpression of LAT1 and 4F2hc Is Necessary to Increase System
L Transport Activity in NIH3T3 Cells--
Other
investigators have demonstrated that overexpression of CD98 heavy
chain/4F2hc transforms NIH3T3 cells and that transformation depends on
association with an unidentified light chain (26, 27). To determine
whether LAT1 transfection can increase transport activity in NIH3T3
cells and to compare activity in hepatocytes versus
fibroblasts, we overexpressed LAT1 alone or with 4F2hc in NIH3T3 cells.
We found that overexpression of both LAT1 and 4F2hc increased system
L-specific transport activity approximately 2-fold
(significant at p < 0.005) relative to LAT1, 4F2hc, or empty vector transfection (Fig.
4a). In contrast to the
studies in hepatocytes, however, overexpression of LAT1 alone had no
significant effect on transport activity. As expected, the addition of
excess cold BCH inhibited leucine transport, which is consistent with system L activity. Arginine transport (system
y+) was not significantly different after any of the
transfections (Fig. 4b). Interestingly, system L
amino acid transport activity was 3-6-fold higher in NIH3T3 cells
compared with AML12 cells on a transport activity per cell basis
(Table I).
Endogenous LAT1 and 4F2hc RNA Levels in AML12 and NIH3T3
Cells--
In previous studies we have seen adaptive regulation of
message for LAT1 but not CD98 heavy chain/4F2 in response to arginine levels in normal but not transformed or tumorigenic hepatic cells (25).
We were interested in whether the response to arginine of LAT1 and
4F2hc message in AML12 and NIH3T3 cells resembled the response of
normal or transformed cells. We also wanted to determine whether
endogenous LAT1 and 4F2hc levels may contribute to the observed
differences in transport activity after transfection between AML12 and
NIH3T3 cells. Immunofluorescence data (above) indicated intense
staining for 4F2hc in both AML12 and NIH3T3 cells and for LAT1 in
NIH3T3 cells but relatively weak staining for LAT1 in AML12 cells.
AML12 and NIH3T3 cells were cultured for 24 h in CEM with or
without arginine. Steady-state LAT1 mRNA levels were very low in
AML12 cells cultured in medium with arginine but induced 5-fold in the
absence of arginine (Fig. 5). Similar results were obtained after 48 h of culture. LAT1 mRNA levels were constitutively high in NIH3T3 cells regardless of arginine availability. Although basal levels of LAT1 message were much higher in
NIH3T3 cells relative to AML12 cells, 4F2hc steady-state message levels
were high and nonresponsive to arginine availability in both cell
types. Under the conditions of the transport assays, i.e. in
the presence of arginine, LAT1 message is low in AML12 cells relative
to NIH3T3 cells, whereas 4F2hc message levels were equivalent in the
two cell types. Thus, message levels correlate well with the observed
LAT1 and 4F2hc immunofluorescence.
Proliferation of LAT1-transfected AML12 Cells Cultured in
Conditions of Limiting Arginine--
LAT1 expression is up-regulated
in nontransformed hepatocytes after culture without arginine, and loss
of that regulation and constitutive expression of LAT1 is associated
with the transformed phenotype (25). We have hypothesized that
up-regulation of LAT1 confers a growth or survival advantage in the
tumor microenvironment. To determine whether AML12 cells overexpressing
LAT1 had a growth advantage in limiting arginine conditions over AML12
cells expressing endogenous LAT1, AML12 cells were transfected with
either pFLAG-LAT1 or pEGFP-F (as a control and monitor of transfection
efficiency). The medium was changed to CEM without arginine immediately
after transfection and changed every 24 h. As shown in Fig.
6, after 48 h of culture AML12 cells
transfected with LAT1 demonstrated approximately a 2-fold increase in
cell number versus GFP-transfected cells after culture
without arginine.
To attempt to elucidate the mechanism for this difference in cell
number, we performed cell cycle analysis on these cells. AML12 cells
transfected with either pFLAG-LAT1 or pEGFP-F were stained with
propidium iodide, and cell cycle analysis was performed at 0, 24, and
48 h post-transfection/switch to CEM without arginine. The results
are presented in Fig. 7. There were no
apparent differences in cell cycle phase distribution after propidium
iodide stain in either cell population at any time examined. The
observed differences in cell number therefore may be due to differences
in rate of proliferation, saturation densities, cell adhesion, or cell
survival.
We examined the functional significance of overexpression of
LAT1/CD98lc and/or 4F2hc/CD98hc in nontransformed hepatic and fibroblast cell lines and found that overexpression of LAT1 alone was
sufficient to increase system L transport activity in mouse hepatocyte but not fibroblast cell lines. Coexpression of LAT1 and
4F2hc in mouse hepatocytes does not increase system L
transport activity above that of LAT1 transfection alone. To our
knowledge, this is the first demonstration of increased transport after
overexpression of a CD98 light chain in the absence of heavy chain.
Exogenous LAT1 presumably complexes with endogenous 4F2hc to mediate
transport. Northern analysis and immunofluorescence suggest that 4F2hc
message and protein are constitutively expressed at nearly equivalent levels in both mouse hepatocyte and fibroblast cell lines. Endogenous steady-state LAT1 message and protein levels, however, are much higher
in mouse fibroblasts versus hepatocytes. Thus, the ratio of
LAT1/4F2hc seems quite different in these cell types. Because NIH3T3
cells express high levels of both LAT1 and 4F2hc, this suggests that
parallel overexpression of these molecules is necessary to elevate
transport still higher. In AML12 cells by contrast, endogenous 4F2hc
levels are high but LAT1 levels low, and overexpression of LAT1 alone
is sufficient to increase transport in AML12 cells.
LAT1 message, but not that of 4F2hc, is responsive to arginine
availability in mouse hepatocytes yet not in fibroblasts. We also found
that basal amino acid transport through both systems L and
y+ was significantly greater in the fibroblast
versus hepatocyte cell lines. The increased transport
activity in NIH3T3 cells may be necessary to support the much higher
growth rate of NIH3T3 versus AML12
cells.2 Our previous studies
have shown that the response of LAT1 and system L transport
to arginine availability is lost gradually in transformed and
tumorigenic hepatic cells. Our results, therefore, may explain the
striking results of Shishido et al. (27), in which only a
2-fold increase in 4F2hc expression was sufficient to transform BALB3T3
cells. They suggest a threshold of CD98-mediated malignant
transformation, and our results are consistent with the notion that
NIH3T3 cells may be close to that threshold. LAT1 response to arginine
in the mouse fibroblasts, although not hepatic cells, is similar to our
observations in transformed, tumorigenic hepatic cells.
In addition to an increase in system L transport, AML12
cells overexpressing LAT1 also displayed an increase in growth under conditions of limited arginine relative to control cells. An enhanced requirement for amino acids has been identified in the nutrient-poor tumor microenvironment (39). The observed difference in proliferation is a potential mechanism whereby LAT1 expression gives neoplastic cells
a growth advantage. Because we did not see any difference in cell cycle
phase distribution in any of the cells at any time during the
proliferation assay, the observed differences in cell number may be
caused by differences in rate of proliferation, saturation densities,
cell adhesion, or cell survival. Further studies with stably expressing
lines or inducible expression constructs are needed to explore the
mechanism(s) involved.
Many factors contribute to neoplastic transformation and malignant
progression including expression of oncogenes, loss of tumor
suppressors, modulation of angiogenic and cell adhesion factors, loss
of cell cycle checkpoint control, etc. There is a growing body of
evidence, however, supporting changes in amino acid transport as also
being a factor in transformation and carcinogenesis, particularly in
the liver. Transport systems L, y+, ASC, and A
show marked differences in activity in normal hepatocytes versus hepatomas (25, 40-43). Changes in hepatic amino acid
metabolism during carcinogenesis include increases in arginine and
glutamine transport in hepatomas versus normal hepatocytes
and increases in total protein synthesis, proportional to tumor burden,
in the livers of tumor-bearing animals (44, 45). Tumor-bearing rats, in
contrast to pair-fed controls, experience an increase in hepatic amino
acid transport via multiple transport systems, perhaps through an
increase in the number of transporter proteins in the plasma membrane
(39). It is possible that the failure to observe increased transport in
AML12 cells with LAT1 and 4F2hc coexpression relative to LAT1 alone may
be caused by a mechanism limiting the number of transporters in the
membrane of these normal epithelial cells.
Inhibitors of cellular proliferation have been used to link the
increased amino acid transport of hepatomas to the increased proliferation rate of these neoplastic cells. Sodium butyrate and
novobiocin inhibited cellular proliferation with a corresponding decrease in leucine, arginine, glutamine, and alanine transport of more
than 50% in fast growing SK-Hep cells in 48 h and of 30 and 50%
in slow growing HepG2 and Huh-7 cells, respectively (44). The more
pronounced inhibitor effect on the faster growing hepatoma is
consistent with greater amino acid transport requirements for faster
growing cells. Changes in amino acid transport may give neoplastic
cells an advantage in metastasizing to new locations as well as an
advantage in establishing a primary tumor. The increased transport
activity of systems A and N in human adenocarcinoma cells, for example,
has been found to enhance the invasiveness of these cells as measured
by an amnion and Amgel invasion assay (46).
We have hypothesized that LAT1 expression is an early event in
hepatocarcinogenesis providing neoplastic cells with a selective growth
or survival advantage, particularly under conditions of nutrient
stress. Our data show that overexpression of LAT1 in nontransformed
hepatocytes can increase amino acid transport and lead to a growth
advantage in conditions of limited amino acid availability. Our data
also suggest that the phenotypic consequences of LAT1 expression can
vary in different cell types. Cells in which LAT1 is not expressed
normally, such as hepatocytes, may acquire a growth advantage if LAT1
becomes constitutively expressed. Studies to examine the mechanisms
contributing to constitutive expression and the phenotypic consequences
of blocked expression in transformed cells both in vitro and
in vivo are planned to establish the relative contribution
of this pathway to tumor growth.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C in the presence of intensifying
screens. Blots were stripped and rehybridized to an 1800-base pair
EcoRI fragment of human 4F2hc (provided by Dr.
Martin Hemler) (32). Densitometry using the Quantity OneTM IBM software
package was used to quantify differences in RNA levels with
normalization to 18 S ribosomal RNA.
4 separate determinations). All data
were normalized for transfection efficiency. Data comparing two
experimental results were analyzed statistically by Student's t test using the InStat Macintosh statistics program. Two
experimental results were considered to be statistically significantly
different when p < 0.05. Each experiment was repeated
with independent transfections at least twice to show qualitatively the
same results. Averages of three independent experiments are shown for
both AML12 and NIH3T3 cells.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(35). This cell
line was established because hepatocytes cannot be maintained for long
periods of time as replicating, differentiated cells while remaining
nontumorigenic. These cells have been used extensively as a model to
study tissue-specific gene regulation and hepatocarcinogenesis because
they are easily transfectable, do not display anchorage-independent
growth or form tumors in nude mice, and maintain expression of
differentiated hepatocyte markers after extensive passaging (35-37).
NIH3T3 cells have been utilized by other investigators (26) to examine
the functional significance of CD98 heavy chain overexpression. AML12 and NIH3T3 cells were cotransfected with pEGFP-F and pFLAG-5a, and
transfection efficiency was monitored 48 h post-transfection by
fluorescence of GFP (Fig. 1).
Transfection efficiencies routinely approached 50 and 40% for AML12
and NIH3T3, respectively. Cotransfection with pEGFP-F was used to
monitor transfection efficiency in all amino acid transport
experiments.
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Fig. 1.
Efficiency of transfection in mouse
cells. NIH3T3 and AML12 cells cotransfected with pEGFP-F and
pFLAG-5a (empty FLAG vector). NIH3T3 cells (a and
b) and AML12 cells (c and d) were
examined 48 h post-transfection under phase and fluorescence
microscopy, respectively. Transfection efficiency was monitored
in all experiments by cotransfection with pEGFP-F.
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Fig. 2.
Immunofluorescence detection and localization
of exogenous and endogenous LAT1 and 4F2hc protein in mouse cells.
A and B, immunofluorescence using antibody to the
epitope tag present in pFLAG-LAT1 or pL(CD98-HA)IZ was performed
48 h after transfection of NIH3T3 (A) or AML12 cells
(B) with each construct. The staining pattern indicated
significant colocalization of LAT1 and 4F2hc protein with intense cell
surface and cytoplasmic staining observed in both cell types.
C, endogenous 4F2hc protein was detected in AML12 and NIH3T3
cells using anti-peptide antibody to 4F2hc. D, endogenous
LAT1 protein was detected in AML12 and NIH3T3 cells using anti-peptide
antibody to LAT1. Intense staining for endogenous 4F2hc was observed at
the cell surface and in the cytoplasm in both cell types. Intense cell
surface and cytoplasmic staining for endogenous LAT1 protein was
observed in the NIH3T3 cells, but in contrast, very weak staining for
LAT1 protein was observed in AML12 cells. The localization of
endogenous and exogenous LAT1 and 4F2hc protein was similar in both
cell types.
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Fig. 3.
Amino acid transport analysis in transfected
mouse hepatocytes. The uptake of 50 µM radiolabeled
leucine and arginine was measured in transfected AML12 cells 48 h
post-transfection with plasmid DNA. All transport values are expressed
as picomoles per mg of protein per min. Transport was found to be
sodium-independent (data not shown), so only transport values in the
absence of sodium are depicted. Each experiment was repeated at least
twice with similar results. Values are means ± S.E. of twelve
measurements from three separate experiments. a, leucine
transport was measured in transfected cells. Excess cold BCH inhibited
leucine transport. b, arginine transport was measured in
transfected cells. Transfection with LAT1 alone significantly increased
leucine transport (p < 0.005) versus
transfection with 4F2hc or empty vector. Cotransfection of LAT1 and
4F2hc did not increase transport above that of LAT1 alone. Arginine
transport did not vary after transfection with any of the
constructs.
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Fig. 4.
Amino acid transport analysis in transfected
mouse fibroblasts. The uptake of 50 µM radiolabeled
leucine and arginine was measured in transfected NIH3T3 cells 48 h
post-transfection with plasmid DNA. All transport values are expressed
as picomoles per mg of protein per min. Transport was found to be
sodium-independent (data not shown), therefore only transport values in
the absence of sodium are depicted. Each experiment was repeated at
least twice with similar results. Values are means ± S.E. of
twelve measurements from three separate experiments. a,
leucine transport was measured in transfected cells. Excess cold BCH
inhibited leucine transport. b, arginine transport was
measured in transfected cells. Cotransfection of LAT1 and 4F2hc
significantly increased leucine transport (p < 0.005)
versus single transfection with LAT1, 4F2hc, or empty
vector. Arginine transport did not vary after transfection with any of
the constructs.
Relative System L amino Acid transport activity in
transfected cells
View larger version (44K):
[in a new window]
Fig. 5.
Expression of CD98lc/LAT1 and CD98hc/4F2hc in
AML12 and NIH3T3 cells in response to arginine availability. Total
RNA was isolated from AML12 and NIH3T3 cells was cultured in CEM with
or without arginine for 24 h. Northern blot hybridization of total
RNA (12 µg) was analyzed by sequential hybridization to probes for
LAT1 (top panel) and 4F2hc (middle panel).
Ethidium bromide staining was used as a loading control between lanes.
Approximate transcript sizes are indicated to the right.
LAT1 but not 4F2hc was induced by arginine deprivation in AML12 but not
in NIH3T3 cells. 4F2hc was constitutively expressed in both cell types.
LAT1 was constitutively expressed at high levels in NIH3T3 cells
relative to AML12 cells.
View larger version (10K):
[in a new window]
Fig. 6.
Proliferation of LAT1 and GFP transfected
AML12 cells cultured without arginine. AML12 cells were
transfected with either pFLAG-LAT1 or pEGFP-F. The cell culture medium
was changed to CEM without arginine immediately after transfection.
Cells were counted by trypan blue dye exclusion at the beginning of the
experiment as well as 24 and 48 h post-transfection/switch to
medium without arginine. At 48 h after culture without arginine,
the population of LAT1-transfected cells was approximately 2-fold
greater (p < 0.005) in cell number than the
GFP-transfected (control) cells.
View larger version (13K):
[in a new window]
Fig. 7.
Cell cycle analysis in transfected AML12
cells after culture without arginine. AML12 cells were transfected
with either pFLAG-LAT1 or pEGFP-F. The cell culture medium was changed
to CEM without arginine immediately after transfection. The cells were
stained with propidium iodide and cell cycle analysis was performed at
0 (A), 24 (B), and 48 (C) h
post-transfection/switch to CEM without arginine. The percentage of
cells in each phase of the cell cycle is shown for each condition.
There were no significant differences in cell cycle phase distribution
after propidium iodide stain in either cell population at any time
examined.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. H. Jauregui for generous gifts
of media, Dr. Suresh Tate for providing -4F2hc antibody, Dr. Nedim
Ince for NIH3T3 cells and helpful discussions, and Dr. Tatiana
Kolesnikova and Dr. Martin Hemler for providing 4F2hc cDNA,
pL(CD98HA)IZ expression construct, and HA.11 antibody as well as
valuable discussions. Dr. Sri Diah and Maggie Medina are
acknowledged gratefully for affinity purification and preliminary
characterization of
-LAT1 peptide antibody.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant CA73611 and NIEHS National Institutes of Health Training Grant T32ESO7272.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Medical
Oncology, Rhode Island Hospital, 593 Eddy St., Providence, RI 02903. Tel.: 401-444-8860; Fax: 401-444-8141; E-mail:
Nancy_Thompson@brown.edu.
Published, JBC Papers in Press, February 22, 2001, DOI 10.1074/jbc.M008248200
2 (W. A. Campbell and N. L. Thompson, unpublished observations)
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ABBREVIATIONS |
---|
The abbreviations used are: CEM, Chee's essential medium; GFP, green fluorescent protein; PBS, phosphate-buffered saline; KRP, Krebs Ringer phosphate; BCH, 2-aminobicyclo-(2, 2, 1)-heptane-2-carboxylic acid; HA, hemagglutinin.
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