From the Departments of Medicine and Molecular
Pharmacology, and the Albert Einstein Comprehensive Cancer Center,
Albert Einstein College of Medicine, Bronx, New York 10461 and
§ Departments of Human Genetics and Pediatrics, Mount
Sinai School of Medicine, New York, New York 10029
Received for publication, August 29, 2000
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ABSTRACT |
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The thiamin transporter encoded by
SLC19A2 and the reduced folate carrier (RFC1) share 40%
homology at the protein level, but the thiamin transporter does not
mediate transport of folates. By using murine leukemia cell lines that
express no, normal, or high levels of RFC1, we demonstrate that RFC1
does not mediate thiamin influx. However, high level RFC1 expression
substantially reduced accumulation of the active thiamin coenzyme,
thiamin pyrophosphate (TPP). This decreased level of TPP, synthesized
intracellularly from imported thiamin, resulted from RFC1-mediated
efflux of TPP. This conclusion was supported by the following
observations. (i) Efflux of intracellular TPP was increased in cells
with high expression of RFC1. (ii) Methotrexate inhibits TPP influx.
(iii) TPP competitively inhibits methotrexate influx. (iv) Loading
cells, which overexpress RFC1 to high levels of methotrexate to inhibit
competitively RFC1-mediated TPP efflux, augment TPP accumulation. (v)
There was an inverse correlation between thiamin accumulation and RFC1
activity in cells grown at a physiological concentration of thiamin.
The modulation of thiamin accumulation by RFC1 in murine leukemia cells
suggests that this carrier may play a role in thiamin homeostasis and
could serve as a modifying factor in thiamin nutritional deficiency as
well as when the high affinity thiamin transporter is mutated.
The reduced folate carrier
(RFC1),1 first cloned in
1994, mediates transport of reduced folates critical to one
carbon-requiring biosynthetic reactions in mammalian cells and is a
member of the major facilitator superfamily of transporters (1-3).
RFC1 also delivers MTX and new generation antifolates into a variety of tumors, particularly those of hematopoietic origin (4). RFC1 exchanges
folates with a broad spectrum of inorganic and organic anions, and high
extracellular concentrations of a variety of organic phosphates
competitively inhibit RFC1-mediated folate influx (5-7). This
interaction between RFC1 and organic phosphates results in the uphill
transport of folates into cells linked to the organic phosphate
gradient across cell membranes (5).
Structurally unrelated to the folates, thiamin plays an essential role
in glycolysis and oxidative decarboxylation reactions after conversion
to the coenzyme thiamin pyrophosphate by thiamin pyrophosphokinase in
cells. Thiamin is also transported across cell membranes by a
carrier-mediated process (8). Thiamin deficiency, reflected in a
decrease in plasma thiamin concentration and TPP levels in
erythrocytes, results in a variety of clinical abnormalities including
cardiovascular and neurological disorders (9). Thiamin deficiency due
to impaired transport results in the thiamin-responsive megaloblastic
anemia syndrome, a disorder also associated with deafness and diabetes
mellitus (10, 11). Positional cloning with families inheriting this
autosomal recessive disease led to the recent identification of the
thiamin transporter gene SLC19A2 (12-14).
The thiamin transporter encoded by SLC19A2 is highly
homologous to RFC1, sharing an amino acid identity of 40% and
similarity of 55%, and both are predicted to have 12 transmembrane
domains. Despite the similarity between these two proteins, the thiamin transporter, when expressed in HeLa cells, was not found to transport folates (15). In the current report, the impact of RFC1 function on
thiamin transport and accumulation of its active coenzyme metabolites was studied in murine leukemia cells. Although RFC1 was not found to
transport thiamin, it does transport phosphorylated thiamin derivatives, thereby modulating the intracellular accumulation of
active thiamin metabolites.
Chemicals--
[3',5',7-3H]MTX (5.7 Ci/mmol) and
[3H]thiamin hydrochloride (20 Ci/mmol) were obtained from
Amersham Pharmacia Biotech, and [3H]TPP (generally
labeled, 4.2 Ci/mmol) was custom-made by Moravek Biochemicals (Brea,
CA). Unlabeled MTX was provided by Lederle Laboratories (Carolina,
Puerto Rico), and thiamin, TMP, and TPP were purchased from Sigma.
Tritiated MTX was purified by high performance liquid chromatography
before use (16), and tritiated thiamin and TPP were used directly after
purity was confirmed by HPLC.
Cells Lines and Culture Conditions--
G1a, G2, D10, and
MTXrA cells were selected from murine leukemia L1210 cells
in the presence of MTX, with or without chemical mutagenesis, as
previously reported (17-21). All four cell lines harbor mutations in
RFC1 resulting in impaired or absent transport mediated by RFC1. Both
R16 and T2 are cell lines with high level expression of RFC1, obtained
by transfecting murine RFC1 cDNA into MTXrA or
wild-type L1210 cells, respectively (21, 22). L7, L15, L44, and L51 are
5,10-dideazatetrahydrofolate-resistant L1210 variants isolated by
chemical mutagenesis followed by selection in the presence of this
drug. In these cell lines folylpolyglutamate synthetase was mutated,
resulting in a marked reduction in activity of the protein, but RFC1
function was not altered (23). All cell lines were grown in RPMI 1640 medium containing 5% bovine calf serum (HyClone), 2 mM
glutamine, 20 µM 2-mercaptoethanol, penicillin (100 units/ml), and streptomycin (100 µg/ml) at 37 °C in a humidified
atmosphere of 5% CO2. For assay of thiamin accumulation,
cells were grown in thiamin-free RPMI medium (custom-made by Life
Technologies, Inc.) containing 5% dialyzed bovine calf serum (Life
Technologies, Inc.), 2 mM glutamine, 20 µM
2-mercaptoethanol, penicillin (100 units/ml), and streptomycin (100 µg/ml) supplemented with 30 nM tritiated thiamin. For
culture of R16 and T2 cells, G418 at a concentration of 750 µg/ml was
included in the medium to ensure stable, high level expression of
RFC1.
Transport Studies--
Cells were harvested, washed twice with
HBS (20 mM HEPES, 140 mM NaCl, 5 mM
KCl, 2 mM MgCl2, 5 mM glucose, pH
7.4), and resuspended in HBS to 1.5 × 107 cells/ml.
For some experiments, glucose-free HBS was also used. Cell suspensions
were incubated at 37 °C for 20 min following which uptake was
initiated by the addition of [3H]thiamin,
[3H]TPP, or [3H]MTX, and samples were taken
at the indicated times. Uptake was terminated by injection of 1 ml of
the cell suspension into 10 ml of ice-cold HBS. Cells were collected by
centrifugation, washed twice with ice-cold HBS, dried, and digested
with 1 N NaOH in an 8-ml vial. After fluor was added,
radioactivity was assessed in a liquid scintillation spectrometer. For
efflux experiments, cells loaded with 0.2 µM
[3H]thiamin for 1 h, were harvested by
centrifugation, washed twice with ice-cold HBS, and resuspended into 9 ml of pre-warmed thiamin-free HBS. Portions were taken, and
intracellular tritium was determined as described as above.
HPLC Analysis--
Cells (~ 8 × 106)
incubated with [3H]thiamin were washed three times with
0 °C HBS. One-fourth of the cell pellet was processed for dry weight
and total tritium as described in transport studies. The remaining
portion was processed according to a slightly modified published
protocol (24). Cell pellets were suspended in 250 µl of HBS, and 15 µl of trichloroacetic acid (100% w/v) was added to precipitate
proteins. After centrifugation the supernatant was extracted with 0.5 ml of water-saturated ether for five times and neutralized with 1 N NaOH. After the residual ether was removed in a
speed-vac, the extract was spiked with unlabeled thiamin, TMP, and TPP
and separated on a reversed-phase HPLC column (Waters Spherisorb, 5 µm ODS2 4.6 × 250 mm) as described previously (25). Separation
of the thiamin, TMP, and TPP was achieved with a linear gradient of
from 0 to 60% of acetonitrile in 70 mM phosphate, pH 7.4, over 30 min followed by a 10-min elution with 70 mM
phosphate, pH 7.4, at a flow rate of 1 ml/min. Under these conditions,
elution times of thiamin, TMP, and TPP were 20, 15.6, and 14 min,
respectively. Fractions (0.5 ml) were collected in 8-ml scintillation
vials, and radioactivity was assessed as indicated above. The levels of
thiamin and its metabolites were normalized to units of nmol/g dry
weight of cells.
Accumulation of Thiamin and TPP in Cells under Growth
Conditions--
Cells (3 × 106) grown in complete
RPMI 1640 were washed twice with thiamin-free RPMI and resuspended into
the same medium supplemented with 30 nM
[3H]thiamin. After 1 week of exponential growth, cells
were harvested, washed twice with ice-cold HBS, and processed for
intracellular tritium as described for transport studies.
The Characteristics of Net Thiamin Uptake and Efflux in Murine
Leukemia Cells That Overexpress, or Lack Functional, RFC1--
Thiamin
uptake was assessed in several murine leukemia cell lines with
different levels of RFC1 function. MTXrA is a subline of
murine leukemia L1210 cells that lack RFC1 activity due to an alanine
to proline substitution at amino acid 130 (21). R16 cells, derived by
transfection of RFC1 cDNA into MTXrA cells (21),
express about 10 times more RFC1 than wild-type L1210 cells. As
indicated in the inset of Fig.
1, MTX uptake in MTXrA cells
was negligible over 30 min, whereas MTX uptake in R16 cells was very
rapid and reached steady state within 10 min (22). The pattern of
uptake of thiamin was reversed in these cell lines. Net uptake in
MTXrA cells was roughly three times greater than that of
R16 cells by 1 h; in neither cell line was a steady state reached
over the interval of observation (upper panel of Fig. 1).
HPLC analysis indicated that at 1 h 90 ± 3 and 75 ± 6% (n = 2) of intracellular tritium was the active
thiamin metabolite, TPP in R16 and MTXrA cells,
respectively; in the latter the remainder of intracellular tritium was
thiamin.
The lower panel of Fig. 1 illustrates the decline in cell
tritium when MTXrA and R16 cells were loaded with 0.2 µM [3H]thiamin for 1 h prior to
resuspension into thiamin-free buffer. The decrease of intracellular
tritium can be characterized by a single exponential in both cell
lines, and the slope of the lines extrapolate through the time 0 points
that represent the initial level of intracellular tritium. The rate
constant for TPP efflux from R16 cells was 4-fold greater than from
MTXrA cells, 0.58 versus 0.14 h Initial Uptake of Thiamin--
Initial thiamin uptake was assessed
in L1210 and R16 cells over 20 s at an extracellular concentration
of 0.2 µM. As indicated in the upper panel of
Fig. 2, the initial uptake rate for
thiamin in R16 cells was ~28% higher than in L1210 cells, much less
than the 9-fold difference in RFC1 expression and MTX influx between these cell lines. Moreover, addition of 25 µM MTX, which
would reduce RFC1-mediated influx by at least 70% (based upon the
RFC1-mediated MTX influx Kt of 7 µM
(17) and a thiamin influx Ki of 2.8 mM,
see below), had no effect at all on thiamin influx in either cell line.
Hence, RFC1 does not contribute to thiamin influx. The very low
affinity of RFC1 for thiamin was confirmed by evaluating the inhibitory
effect of this vitamin on MTX influx in L1210 cells. As shown in the
lower panel of Fig. 2, there was a gradual increasing
inhibition of MTX influx, albeit to a small degree, as the thiamin
concentration was increased from 0.1 to 1 mM. At 1 mM thiamin, MTX influx was reduced by only ~28%. Based upon the MTX influx Kt of 7 µM, the
Ki for thiamin was calculated to be 2.8 mM.
TPP Influx in Murine Leukemia Cells--
Since thiamin is rapidly
phosphorylated to TPP in these cells, one explanation for low net
thiamin uptake in cells with high level RFC1 expression (R16) is that
TPP generated in the cell is a substrate for, and is exported by, RFC1.
To explore this possibility, the effect of TPP on MTX influx was
assessed along with the transport properties of TPP (Fig.
3). TPP inhibited MTX influx with a
Ki of 32 ± 5 µM
(n = 3) in L1210 cells (upper panel), a
value only ~4-fold higher than the MTX influx Kt
of 7 µM, consistent with a previous report (26). Furthermore, TPP influx was directly related to the level of RFC1 activity. Influx in the MTXrA cells was one-fourth that of
wild-type L1210 cells, and influx was 7-fold greater in R16 cells than
L1210 cells. Furthermore, 25 µM MTX markedly decreased
TPP influx in R16 and L1210 cells, whereas TPP initial uptake in
MTXrA cells was the same in the presence or absence of 25 µM MTX (lower panel). Hence, RFC1-mediated
influx of TPP is equal to, or larger than, transport mediated by other
process(es).
The Effect of Intracellular MTX on Net Thiamin Uptake--
If TPP
efflux is indeed mediated by RFC1, loading cells to high levels of MTX
should competitively inhibit efflux of TPP and augment net TTP cellular
accumulation. This was found to be the case. Incubation of R16 and
MTXrA cells with 1 mM MTX resulted in
intracellular MTX levels of 350 and 220 nmol/g dry wt (100 and 63 µM, respectively, based upon a ratio of intracellular
water to dry weight of 3.5 µl/mg), since MTX enters cells via passive
diffusion and possibly routes other than RFC1 at this high
concentration. As shown in Fig. 4, intracellular accumulation of thiamin and its metabolites was doubled
in R16 cells, to a level comparable to that of MTXrA cells,
by pre- and co-incubation with 1 mM MTX. HPLC analysis confirmed that this increase in net uptake was due entirely to an
increase in TPP accumulation. On the other hand, there was no effect of
1 mM MTX on thiamin uptake in MTXrA cells, all
consistent with the lack of RFC1 function in this cell line.
Energy Dependence of Net Thiamin Uptake--
When L1210,
MTXrA, and R16 cells are incubated with 10 mM
azide in the absence of glucose, intracellular ATP is depleted, an effect at least partially reversed by addition of 5 mM
glucose to the transport buffer (5). As indicated in Fig.
5 (upper panel), net thiamin
uptake in MTXrA cells was higher than that in L1210 cells
without energy depletion. However, thiamin uptake in ATP-depleted cells
was decreased to the same level in both L1210 and MTXrA
cells. Net thiamin uptake in R16 cells was markedly lower than in L1210
and MTXrA cells regardless of the energy status. This
energy dependence of thiamin accumulation was further explored in L1210
cells by determination of thiamin metabolites by HPLC (lower
panel of Fig. 5). Under all conditions, TMP was present at levels
that were barely detectable as compared with thiamin and TPP. In
energy-depleted cells, the level of thiamin exceeded that for TPP
regardless of the time of exposure to tritiated thiamin (10 or 60 min),
but some TPP nonetheless was present. There were slightly higher levels of both thiamin and TPP at 1 h of exposure than that at 10 min reflecting continued, albeit slow, thiamin uptake and phosphorylation. In contrast, TPP was the dominant species in energy-replete cells. The
increase in TPP accumulation from 10 min to 1 h contributed almost
entirely to the increase in net thiamin uptake in L1210 cells. The
lower level of thiamin present in energy-replete, versus energy-deplete, cells may be due to the rapid rate of phosphorylation relative to the rate of thiamin entry into cells.
The Impact of RFC1 Function on Thiamin Accumulation at a
Physiological Thiamin Concentration--
In most media, including RPMI
1640, in which murine leukemia cells are grown, the thiamin
concentration (~3 µM) is about 2 orders of magnitude
higher than the physiological blood level (10-30 nM).
Hence, in vitro growth conditions are highly
nonphysiological with respect to this vitamin. To determine the effect
of RFC1 function on thiamin accumulation under more physiological
conditions, cells were grown in 30 nM
[3H]thiamin for 1 week. In addition to wild-type L1210,
MTXrA, and R16 cells, another eight L1210 variants were
studied in these experiments. These included the following: (i)
L1210-T2 cells obtained by transfection of RFC1 cDNA into wild-type
L1210 cells to achieve RFC1 expression at a level about 7-fold higher than in L1210 cells (22); (ii) L7, L15, L44, and L51 cell lines with
functional RFC1 but with markedly reduced folylpolyglutamate synthetase
activity due to mutations in this enzyme (23); (iii) the D10 line with
no expression of RFC1 due to a Gly to Ala substitution in the
initiation codon (19); and (iv) G1 and G2 cells with point mutations
resulted in S46N and V104M substitutions, respectively, that markedly
impair RFC1 activity (17, 18). As indicated in Table
I, thiamin accumulation generally
correlated with levels of RFC1 activity. Thiamin accumulation in R16
and T2 cells was 64 and 42%, respectively, that of L1210 cells,
whereas thiamin accumulation in L7, L15, L44, and L51 cells, in which
RFC1 function is near normal, was comparable to that of L1210 cells.
However, thiamin accumulation in G1a, G2, MTXrA, and D10
cells with markedly impaired RFC1 function was increased by factors of
1.8, 2.4, 2.8, and 2.5, respectively. On average, the level of thiamin
and its metabolites in cells that overexpress RFC1 (R16 and T2) was
4-7-fold greater than in cells in which RFC1 function is absent
(MTXrA and D10).
Thiamin transport in murine leukemia cells is likely mediated by a
facilitative carrier. Accumulation of its active coenzyme form is
attributed to rapid phosphorylation of thiamin to TPP which is, to a
large extent, retained within cells. This is a common phenomenon for
many different substrates of the major facilitator superfamily, as
occurs with phosphorylation of nucleosides (27, 28) and polyglutamation
of folates (29). Consistent with this was the observation that in
energy-depleted cells, phosphorylation was impaired and net thiamin
uptake was markedly decreased (Fig. 5). Interestingly, the steady-state
thiamin level in energy-depleted L1210 cells was ~0.3
µM (1 nmol/g dry weight), comparable to the extracellular
level (0.2 µM), consistent with an equilibrating process.
Folate and thiamin, both B family vitamins, differ not only in chemical
structure but also in charge. At physiological pH, folate is a bivalent
anion, whereas thiamin bears one positive charge. The similarity
between RFC1 and the thiamin transporter, especially in the predicted
transmembrane domains, raised the possibility that these carriers may
share common substrates. This was not the case. Instead, we have
demonstrated that RFC1 transports the major thiamin metabolite, TPP,
with an influx Ki only ~4 times greater than the
MTX influx Kt. This has important consequences with
respect to the level of TTP accumulation. After thiamin enters cells it
is phosphorylated to TPP and its molecular charge changes from positive
to negative. This, in turn, is associated with an increased affinity
for RFC1, an anion exchanger, that mediates TPP efflux, leading to a
decrease in net intracellular TPP accumulation. Accordingly, TPP
accumulation in these cells is enhanced by the rate of entry of thiamin
mediated by the thiamin transporter and the rate of phosphorylation
catalyzed by thiamin pyrophosphokinase. TPP accumulation is countered
by the efflux of TPP mediated by RFC1. Hence, the net level is
determined by balance of these processes. Since efflux studies show
that the major portion of intracellular TPP is retained within the
cells, there must be a large component that is bound to
TPP-dependent apoenzymes in cytosol and mitochondria (30).
In addition, TPP may be hydrolyzed, in part, to TMP which is, in turn,
exported. TMP also appears to be a good substrate for RFC1 since TMP
inhibits RFC1-mediated MTX influx with a Ki of 15 µM.2
Increased thiamin accumulation associated with decreased RFC1 activity
observed when cells were grown at a physiological thiamin concentration
(~30 nM) suggests that the ability of RFC1 to export TPP
may have important biological consequences. The requirement for thiamin
is ubiquitous, but thiamin-responsive megaloblastic anemia syndrome and
dietary thiamin deficiency cause tissue-specific and nonoverlapping
defects (9-11). Metabolically active tissues are vulnerable to thiamin
deficiency due to heavy usage of TPP-dependent enzymes. The
observation that RFC1 mediates efflux of TPP may provide another
important dimension to the understanding of thiamin metabolism. Tissues
with high expression of RFC1 may export more TPP, making them more
susceptible to metabolic derangement when thiamin is scarce. Hence,
RFC1 expression may modulate the tolerance to thiamin deficiency
associated with either mutations in SLC19A2, dietary
deficiency, or malabsorption. It remains to be established if RFC1 is
expressed in thiamin-sensitive cell types and whether metabolic defects
associated with thiamin deficiency might be modified by the level of
RFC1 expression.
Although TPP is not present in plasma, but accumulates in erythrocytes,
TMP is present in plasma at concentrations only slightly lower than
that of thiamin (31). As indicated above, TMP is likely a good
substrate for RFC1, and transport by this route could be of importance
under conditions in which the thiamin transporter is defective. Hence,
at high blood levels of thiamin and TMP, substantial delivery of TMP
via RFC1 might obviate the consequences of the loss of the thiamin
transporter, another potential role for RFC1 as a modifying element in
this clinical situation.
RFC1-mediated TPP transport could also play a role in thiamin
absorption in intestine. Dietary vitamin B1 exists
predominantly as thiamin pyrophosphate that is hydrolyzed to thiamin by
a phosphatase in the intestinal lumen before absorption. RFC1 is
expressed in intestine and is proposed to mediate folate absorption
(32, 33). It is possible that some TPP may be directly delivered into
mucosal cells by RFC1 before hydrolysis to thiamin is achieved. Furthermore, TMP is derived from dephosphorylation of TPP and/or transphosphorylation of thiamin by a membrane-associated alkaline phosphatase in intestinal mucosa (34, 35). TMP has been shown to cross
everted rat jejunal sac wall unchanged and enter the serosal fluid
(36). It is possible that this process is
RFC1-dependent.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Time course of net thiamin or MTX uptake and
efflux in MTXrA and R16 cells. Upper panel,
after a 20-min incubation in HBS at 37 °C, cells were exposed to 0.2 µM [3H]thiamin. Data are the mean ± S.E. of three independent experiments. Inset, representative
MTX uptake at 1 µM, in MTXrA and R16 cells.
Bottom panel, cells exposed to 0.2 µM
[3H]thiamin for 1 h were harvested, washed twice
with cold HBS, resuspended into a large volume of thiamin-free HBS, and
intracellular tritium determined. Data are the mean ± S.E. of
three separate experiments. When not apparent, error bars
are smaller than the symbols.
1, respectively. However, in both cell lines
the rate of loss of TPP was only a small fraction of the rate of
thiamin influx (see below).
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Fig. 2.
The effect of MTX on thiamin influx in L1210
and R16 cells (upper panel) and the effect of thiamin
on MTX influx in L1210 cells (bottom panel).
Upper panel, L1210 and R16 cells were incubated in HBS at
37 °C for 20 min and then exposed to 0.2 µM
[3H]thiamin in the absence or presence of 25 µM MTX. The data are the average of three separate
experiment ± S.E. Bottom panel, after a 20-min
incubation in HBS at 37 °C, 1 µM [3H]MTX
was added in the absence or presence of 0.1, 0.3, or 1 mM
thiamin. Data are the composite of three separate experiments ± S.E. When not apparent, error bars are smaller than the
symbols.
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Fig. 3.
Dixon plot of TPP inhibition of MTX influx
(upper panel) and MTX inhibition of TPP influx in
MTXrA, L1210, and R16 cells (bottom
panel). Upper panel, after L1210 cells were
incubated in HBS at 37 °C for 20 min, tritiated MTX at
concentrations of 1 (triangles) or 2 µM
(squares) was added to the suspension along with TPP at the
concentrations indicated, and incubation was continued for 4 and 2 min,
respectively. Bottom panel, after a 20-min incubation, R16
(inverted triangles), L1210 (squares), and
MTXrA (circles) cells were exposed to 1 µM tritiated TPP in the absence (closed
symbols) or presence (open symbols) of 25 µM unlabeled MTX. For both panels, data shown are
representative of three separate experiments.
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Fig. 4.
Effect of 1 mM MTX on net thiamin
uptake in R16 and MTXrA cells. R16 and
MTXrA cells were first incubated in HBS for 5 min and then
exposed to 1 mM unlabeled MTX for 15 min before 0.2 µM tritiated thiamin was added to the cell suspension. In
control experiments no MTX was added. Data are the means ± S.E.
of three separate experiments.
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Fig. 5.
Effect of energy status on net thiamin uptake
in MTXrA, L1210, and R16 cells (upper
panel) and relative levels of intracellular thiamin and TPP
in L1210 cells (lower panel). Upper
panel, MTXrA, L1210, and R16 cells were incubated in
HBS or glucose free-HBS at 37 °C for 5 min before 10 mM
azide was introduced into the cell suspensions. After an additional
15-min interval, 0.2 µM tritiated thiamin was added to
initiate transport. Energy+ indicates cells resuspended into
HBS containing 5 mM glucose, and energy
indicates cells resuspended in glucose-free HBS. Data are the
means ± S.E. of three experiments. When not apparent, error
bars are smaller than the symbols. Bottom panel, L1210
cells were exposed to 0.2 µM tritiated thiamin for 10 min
or 1 h under the same conditions as described in the upper
panel either in HBS or glucose free-HBS. Following this,
intracellular thiamin and its metabolites were extracted and analyzed
by HPLC. Data are the average of two separate experiments ± S.E.
Accumulation of thiamine in murine leukemia cell lines
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Laibin Liu for technical assistance.
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FOOTNOTES |
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* This work was supported by NCI Grants CA-39807 and CA-82621 (to I. D. G) from the National Institutes of Health and the March of Dimes Basic Science Award 6-FY00-283 (to B. D. G).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: Albert Einstein College of Medicine Comprehensive Cancer Research Center, Chanin 2, 1300 Morris Park Ave., Bronx, NY10461. Tel.: 718-430-2302; Fax: 718-430-8550; E-mail: igoldman@aecom.yu.edu.
Published, JBC Papers in Press, October 18, 2000, DOI 10.1074/jbc.M007919200
2 R. Zhao, F. Gao, Y. Wang, G. A. Diaz, B. D. Gelb, and I. D. Goldman, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: RFC1, the reduced folate carrier; HBS, HEPES-buffered saline; MTX, methotrexate; SLC19A2, the thiamin transporter gene; TMP, thiamin monophosphate; TPP, thiamin pyrophosphate; HPLC, high pressure liquid chromatography.
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REFERENCES |
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