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(Received for publication, June 12, 1995; and in revised form, September
12, 1995) From the
L1210 cell variants selected in the presence of the lipophilic
dihydrofolate reductase inhibitor, metoprine, expressed increased
levels of one-carbon, reduced folate transport inward (Sirotnak, F. M.,
Moccio, D. M., and Yang, C.-H.(1984) J. Biol. Chem. 259,
13139-13144). Growth of one of these variants (L1210/R69), with
metoprine in the presence of decreasing concentrations of
l,L5-CHO-folateH
Cellular folates exist primarily as The process of
folylpolyglutamylation in normal proliferative and neoplastic mammalian
tissues is important (1, 2, 3, 4, 5) to the
conservation and efficient utility of folate coenzymes that are
required for one-carbon transfer reactions during macromolecular
biosynthesis. Consequently, levels of FPGS activity appear to be
highest in the proliferative fraction of normal differentiating
tissues(7, 17, 18, 19) . It has been
suggested in the context of earlier reports (for review, see (20, 21, 22, 23) ) that normal
proliferative and tumor cells might control their macromolecular
synthesis through regulation of intracellular folate homeostasis. In
addition to the biosynthesis and metabolic interconversion of these
compounds(20, 21) , folate homeostasis could also be
regulated at the level of mediated entry of exogenous folate (23) and/or through biosynthesis of
folylpolyglutamates(1, 2, 3, 4, 5) .
With the recent derivation (24) of the cDNA for human FPGS,
studies addressing this issue at the level of FPGS gene expression and
its regulation are now possible. Toward this objective, we now describe
studies with a novel group of metoprine-resistant variants of the L1210
cell that were further characterized and found to constitutively
overproduce FPGS to varying extent. These variant cell lines, which
also overproduce the reduced folate
transporter(25, 26) , were selected during growth in
the presence of this lipophilic antifolate and decreasing amounts of
l,L5-CHO-folateH
The
3- and 8-fold increase in FPGS activity observed in the
metoprine-resistant variants when compared with the parental cells was
accounted for by (Fig. 1) a commensurate increase in values for V
Figure 1:
Kinetic analysis of FPGS activity in
variant and parental cells selected for resistance to metoprine or MTX.
The experimental details are given in the text. The data are derived
from measurements of FPGS activity normalized with respect to protein (v = pmol/min/mg of protein) in cell-free extract at
different concentrations of aminopterin. Average of three experiments
done on different days ± S.E. <
±12%.
Figure 2:
Immunoblotting of FPGS in variant and
parental cells with anti-FPGS peptide antibody. Forty µg (A) or 100 µg (B) of sample of partially purified
cell-free extract was solubilized in SDS-polyacrylamide gel
electrophoresis sample buffer and electrophoresed(39) .
Additional experimental details pertaining to the sample preparation
and the Western blotting are given in the text. The data shown in A and B are for a separate blot following electrophoresis
done under different conditions.
Figure 3:
Northern blot analysis of FPGS poly(A)
+ mRNA from parental and variant L1210 cells with either increased (L1210/R83 and L1210/R84) or decreased (L1210/R25) FPGS activity. Cells were cultured in the
appropriate medium, removed by centrifugation and washed once in
phosphate-buffered saline prior to extraction of mRNA. Aliquots of 5
µg of each mRNA preparation were added to gels for Northern
blotting, electrophoresed, and probed with
Since differences in stability of FPGS
mRNA may be the explanation for the differences in its level among
these variants, we employed the same methodology to determine the
stability of FPGS mRNA in these variants compared with parental L1210
cells. In the experiment shown (Fig. 4), L1210/R84 and parental
cells were exposed to actinomycin D during growth in culture, and
aliquots of cells were removed after varying periods of time for
Northern blotting with FPGS and
Figure 4:
Northern blot analysis of the decay of
FPGS poly(A) + mRNA from actinomycin D-treated parental and
variant L1210 cells with increased (L1210/R84) FPGS activity.
Cells were grown in the presence of 5 µg/ml actinomycin D, and
aliquots of the cell suspension were removed at various time intervals
for mRNA extraction. Additional experimental details are provided in
the text and in the legend of Fig. 3. The data in A represent one of a typical series of blots of FPGS mRNA controlled
for
Figure 5:
Nuclear run-on analysis of FPGS mRNA
transcription in parental and variant L1210 cells with either increased (L1210/R84) or decreased (L1210/R25) FPGS activity.
Similar to our studies of one-carbon reduced folate
transport(25, 26) , the data presented here appear to
document a contrasting role for FPGS as a determinant of resistance to
these two categories of folate antagonists. Elevated levels of FPGS
activity are exhibited by metoprine-resistant L1210 cells, while FPGS
levels are reduced in the MTX-resistant variant also examined in these
studies. Within each category of resistant cells, the basis for these
alterations appears to reflect different levels of FPGS gene
expression, specifically, the rate of FPGS mRNA transcription. We have
previously documented (49) lower levels of FPGS activity and of
folate analogue polyglutamate formation in another group of L1210 cell
variants resistant to a new classical folate analogue, edatrexate. The
molecular basis for these alterations has yet to be elucidated.
However, in contrast to the current results, no alteration was found (50) in preliminary studies on the level of FPGS mRNA in any of
these variants. Downward alterations of FPGS activity in variants ( (14) and (15) , and this study) resistant to classical
antifolates further substantiate the importance of polyglutamylation to
their mechanism of action and the role of FPGS in addition to
one-carbon reduced folate transport as determinants of cytotoxicity to
these agents. In the case of the lipophilic antifolate, metoprine,
resistance appears to be engendered by an increase in both folate
transport inward and FPGS activity as it pertains to natural folate
compounds. In contrast to classical antifolates, neither property is
involved in the internalization or metabolic disposition of this agent
in tumor cells. However, because of their role (1, 2, 3, 4, 5) in
maintaining intracellular levels of folate coenzyme polyglutamates,
increased expression of these properties apparently renders the cell
less sensitive to the folate antagonism mediated by this lipophilic
antifolate. In support of this notion, other data also show (Table 1) that altered levels of expression of FPGS in addition
to folate transport inward profoundly modulate the requirement for
exogenous folate during growth of these cells in culture. Such effects
indicate a role for both properties in maintaining folate homeostasis
in these tumor cells. Although we sought to select for variants with
increased FPGS activity by reducing the folate content of the medium in
the presence of a fixed level of metoprine, these variants do exhibit
increased resistance to metoprine. Therefore, it would be expected in
view of the above considerations that derivation of similar variants
with elevated levels of FPGS would also occur by selection in
increasing levels of metoprine alone.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
U33557[GenBank].
Volume 270,
Number 45,
Issue of November 10, 1995 pp. 26918-26922
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
(natural diastereoisomer of
5-formyltetrahydrofolate), resulted in the selection of other variants
(L1210/R82, R83, and R84) with further reduction in one-carbon, reduced
folate transport and in two cases (L1210/R83 and R84) with
3-8-fold increased folylpolyglutamate synthetase (FPGS) activity
and folate compound polyglutamate formation in situ. Metoprine
resistance was further increased, and the requirement for exogenous
folate during growth was decreased as well in these variants. The
increase in FPGS activity observed in L1210/R83 and R84 was
characterized by 3- and 8-fold increases in value for V
with no change in K and the same increase in a 60-61-kDa protein as shown
by immunoblotting. Northern blotting revealed the same increases in
these two variants in the level of a 2.3-kilobase FPGS mRNA when
compared with control, while Southern blotting of genomic DNA did not
reveal any increase in FPGS gene-copy number or restriction
polymorphisms. Also, no difference in stability of FPGS mRNA was found
between parental and variant cells. In contrast, nuclear run-on assays
revealed differences among these cell types in the rate of FPGS mRNA
transcription that correlated with increased FPGS activity, protein,
and mRNA level in the variants. Similar studies with a
transport-defective, methotrexate-resistant L1210 cell variant
(L1210/R25) documented a 2-3-fold decrease in FPGS activity,
protein, and mRNA levels that was accounted for by a decrease in FPGS
mRNA transcription. These results provide the first examples of
constituitively altered transcriptional regulation of FPGS activity
associated with acquired resistance to antifolates.
-polyglutamate peptides (1, 2, 3, 4, 5) of varying
chain length. Their metabolism to polyglutamates and that of folate
analogues are mediated (1, 2, 3, 4, 5) by the
enzyme, folylpolyglutamate synthetase (FPGS), (
)and
metabolic turnover of these anabolites appears to be modulated by
folylpolyglutamate hydrolase after their mediated entry into lysosomes
(for review, see (6) ). In tumors and normal proliferative
tissues of animals and man, the process of polyglutamylation has
pharmacologic relevance with respect to the cytotoxicity (8, 9) and therapeutic
utility(7, 8, 9, 10, 11, 12, 13) of classical folate analogues. Also, both decreased levels
of FPGS activity (14, 15) and increased levels of
folylpolyglutamate hydrolase activity (16) have been associated
with acquired resistance to these analogues. The mechanistic basis for
these alterations remain to be elucidated. that was increasingly growth limiting. For
comparison, we also describe a methotrexate-resistant L1210 cell
variant, which in addition to markedly reduced transport inward (26, 27) of folate compounds exhibits lower levels of
FPGS activity compared with parental cells. Our results document, as
the molecular basis for the altered level of FPGS activity in all of
these variants, a constitutive increase or decrease in the rate of FPGS
mRNA transcription depending upon the antifolate in question. These
results are described in detail below.
Cells and Culture Conditions
Methods for the
isolation of variant L1210 cells with elevated one-carbon reduced
folate transport and FPGS activity were similar to those reported
earlier (25, 26, 27, 28, 29) from
this laboratory. L1210 cell variants were first selected during growth
in the presence of increasing amounts of metoprine (IC = 77 nM) in folate-free RPMI medium supplemented
with dialyzed fetal calf serum and 20 nM l,L5-CHO-folateH
, which allowed maximum growth in
drug-free medium. Later selection steps utilized growth in 600 nM metoprine and decreasing amounts of l,LCHO-folateH as
the sole folate source in order to avoid the selection of variants with
elevated levels of dihydrofolate reductase. At each step in the
selection, the variant was cloned by limiting dilution and was examined
for [
H]MTX transport inward (25, 26, 27, 28, 29) and
FPGS activity. Subsequent selection steps were carried out with the
cloned subline. Requirements for l,L5CHO-folateH
(EC) and inhibition by folate antagonists
(IC
) were determined by methods described
previously(8, 27) .
Assay for FPGS activity
After processing of
cells(2, 14) grown in drug-free medium, the protein
concentration of the resulting cell-free extract was
determined(30, 31) , and the assay for FPGS activity
was carried out as described previously (2, 13, 14) using aminopterin as substrate.
Product formation (aminopterin + GI) under these conditions was
linear for at least 2 h at 37 °C, and these cell-free preparations
under the assay conditions exhibited (12) no detectable
folylpolyglutamate hydrolase activity.Derivation of a Murine FPGS cDNA Probe
A murine
FPGS cDNA (ZAP-L1210/R83-1) was obtained by hybridization
screening (32) of an L1210 cell cDNA library in gt11
(Stratagene, La Jolla, CA) using a human cDNA, pTZ 18U(24) , as
a probe. This
gt11 cDNA construct incorporates a 2.296-kilobase
insert ligated at the XhoI polylinker site including 3`- and
5`-untranslated region sequences of 461 and 74 base pairs,
respectively, and an open reading frame of 1761 base pairs, which codes
for a putative mitochondrial leader peptide as well as the enzyme
protein. The homology exhibited (data not shown) between the murine and
human cDNAs was 79% at the level of the nucleotide sequence. Sequencing
of the murine cDNA was by the method of Sanger et al.(33) .
Northern Blot Analysis of FPGS mRNA
A method (34) utilizing rapid isolation of poly(A) RNA
directly from the cell lysates by means of an oligo(dT) column was
used. The integrity of the RNA was assessed by electrophoresis in 1.1%
agarose containing 1 M glyoxal and staining with ethidium
bromide. An aliquot (2-10 µg) of the same RNA was analyzed (35, 36) by Northern blotting and radioautography
using a murine FPGS cDNA, ZAP L1210/R83-1 (see above), as a probe
and normalized to mRNA content with a human
-actin probe,
PCD-
-actin(37) . Labeling of each probe was by random
priming (Random Primers DNA labeling kit, Boehringer Mannheim) using
[
-P]dCTP (3000 Ci/mmol and 10 ng of
insert). Direct measurement of total radioactivity in each blot minus
background was obtained with a Betagen 603 blot analyzer.
Southern Blot Analysis
Genomic DNA from parental
and resistant cells as a frozen pellet was prepared (38) and
digested with EcoRI and HindIII. The DNA was
separated by electrophoresis through a 0.82% agarose gel and
transferred (38) to Nytran (Schleicher and
Schuell). DNA probes, hybridization and labeling procedures were the
same as that used above.
Immunoblotting Procedure
Samples of a 0-30%
ammonium sulfate fraction (13) of cytosol were solubilized in
SDS sample buffer and electrophoresed (39) on a 7.5%
polyacrylamide slab gel and transferred to nitrocellulose using a
Bio-Rad Trans-Blot cell(40) . Western blotting was performed (40, 41) using anti-human FPGS peptide polyclonal
antibody (0.5 µg/ml) prepared in rabbits and purified on a
peptide-Sepharose column(41, 42) . The human FPGS
peptide utilized was deduced from the cDNA nucleotide sequence (24) beginning at Val and ending at
Trp
in the order
Val-Val-Cys-Gly-Val-Ser-Leu-Gly-Ile-Asp-His-Thr-Ser-Ser-Leu-Leu-Gly-Asp-Thr-Val-Glu-Lys-Ile-Ala-Trp.
The peptide was linked by EDC-mediated coupling (43) to keyhole
lympet hemacyanin for monthly injections into a rabbit. During
blotting, the anti-rabbit horseradish peroxidase-IgG conjugate (Sigma)
at a 1:3000 dilution was used as the secondary antibody. The blots were
used to expose hyperfilm after incubation with ECL reagents (Amersham
Corp.) and quantitated by scanning densitometry (Stratagene).
Nuclear Run-on Assay
These assays were performed
with nuclear extracts essentially as described in earlier
reports(44, 45) . The cDNA probes used in the blot for
relative transcript level were murine FPGS (see above) and murine
dihydrofolate reductase(46) . cDNA probes for 
- (47) and -actin (37) were used as a positive
control. A cDNA probe for plasmid-derived neomycin resistance (48) was used as a negative control.
Materials and Other Analytical
Methods
[H]MTX
(3`,5`,9`-[H]MTX) (specific activity, 22 Ci/mmol)
was purchased from Moravek Biochemicals, City of Industry, CA.
Aminopterin was a generous gift of Dr. J. R. Piper of the Southern
Research Institute. These materials were repurified by HPLC (8) to >97% purity. Polyglutamates of
[H]MTX were measured in cell extracts by
analytical HPLC(8) . Folyl-polyglutamate hydrolase activity was
measured as described previously(12) .
Isolation and Biochemical Characterization of the
Variant Cell Lines
All of the variant L1210 cell lines used in
these studies were derived from a metoprine-resistant variant
(L1210/R69), which was selected (25) for growth in the presence
of 600 nM metoprine and 20 nM l,L5CHO-folateH as the sole folate source. These additional variants were
selected from L1210/R69 in a stepwise manner following growth in 600
nM metoprine and 4 nM (L1210/R82), then 2 nM
(L1210/R83), and finally 1 nM (L1210/R84)
l,L5CHO-folateH with cloning of the variant after each
selection step. The properties of L1210/R69 and its antecedents were
described earlier(25) . The requirement for
l,L5-CHO-folateH for growth by these newly cloned variants
in the presence of 600 nM metoprine was determined (Table 1) and found to be progressively lower than for parental
cells with each additional selection step. Furthermore, resistance to
metoprine increased so that the relative requirement for this folate
among these variants (Table 1) was inversely related to their
resistance to metoprine. Two biochemical alterations were documented in
these variants, which appeared to allow their growth in the presence of
increasing concentrations of metoprine and/or decreasing concentrations
of folate when compared with parental L1210 cells. At the initial
stages of selection ( (25) and Table 1), one of the
variants derived exhibited elevated transport inward of folate
compounds that was directly related to resistance and inversely related
to the folate requirement. At later stages of selection, in addition to
a further increase in folate transport inward, two of these variants
exhibited (Table 1) a 3- and 8-fold increase in FPGS activity
that was also directly related to resistance and inversely related to
the folate requirement. All three variants were collaterally sensitive
to methotrexate (data not shown). In every case (Table 1), the
folate requirement in the presence of metoprine and the relative
resistance to this lipophilic antifolate of the variants when compared
with parental cells could be accounted for by the alterations both at
the level of folate compound transport inward and of FPGS activity that
were observed when smaller changes (Table 1) in the relative
dihydrofolate reductase activity were also taken into account. We also
provide data in this table (Table 1) on a methotrexate resistant
variant (L1210/R25) for comparison. This cell line exhibited markedly
lower folate compound influx and almost 3-fold lower FPGS activity.
Moreover, it will not grow in l,L5-CHO-folateH as the sole
folate source and is highly collaterally sensitive to metoprine.
for variant FPGS activity with no change in
value for apparent K
. Western blotting was carried
out after SDS-polyacrylamide gel electrophoresis of partially purified
FPGS from cell-free extract from variant and parental L1210 cells (Fig. 2A) with anti-FPGS peptide antibody. Densitometry
(data not shown) revealed the same relative increases (3-8-fold)
among these variants in the amount of a 60-61-kDa protein. Both
results taken together suggested that the increase in FPGS activity
observed in these variants resulted from an elevation in level of FPGS
enzyme protein. In contrast, the V for FPGS
activity in L1210/R25 cells was reduced almost 3-fold (Fig. 1),
and SDS-polyacrylamide gel electrophoresis and Western blotting with
densitometry of partially purified cell-free extract detected (Fig. 2B) 2-3-fold less of a 60-61-kDa
protein. The total difference in FPGS activity among all of these
variants was 15-20-fold. This difference and that for folate
transport inward was reflected (data not shown) in the relative amount
of total intracellular polyglutamates of [H]MTX,
used as a model folate compound, that were found in these various cell
types when grown in the presence of this folate analogue.
Relative Level and Stability of FPGS Poly(A)
Northern blotting of FPGS mRNA from variant and
parental cells revealed (Fig. 3) substantial differences among
these cell types. FPGS mRNA levels, shown in the figure, when compared
with parental cells, were increased (Fig. 3A) 3-fold in
L1210/R83 cells and 7-8-fold in L1210/R84 cells when blots were
normalized during Betagen blot analysis (data not shown) with respect
to mRNA among Variant and Parental L1210
Cells
-actin mRNA level used as a control. In contrast, the level of
FPGS mRNA in L1210/R25 cells was decreased 2-3-fold (Fig. 3B) when related in the same way to the same
-actin mRNA control.
P-labeled ZAP
L1210/R83-1 after transblotting. Additional experimental details
are provided in the text. The figure shows one of several separate
blots of FPGS mRNA from L1210, L1210/R83, and L1210/R84 (A)
and L1210 and L1210/R25 (B) controlled for
-actin mRNA
and done under different conditions. The
-actin mRNA blot was
arbitrarily positioned in the figure with respect to the FPGS mRNA blot
in each case.
-actin cDNA. The results of a
typical experiment (Fig. 4A) show that the rate of
decay of FPGS mRNA with time was essentially the same for each cell
line. The radioactivity in each blot was also determined for replicate
experiments by a Betagen blot analyzer, and the average results for
FPGS mRNA normalized against
-actin mRNA are given in Fig. 4B. These data allowed the quantitation of the
half-time for FPGS mRNA decay from each cell type, which was found to
be the same (half-time = 5.8 ± 0.8 h). Stability of
L1210/R25 FPGS mRNA was also determined (data not shown) in the same
way and found to be the same as parental cell and L1210/R84 cell FPGS
mRNA.
-actin mRNA. The data in B are from an analysis of
radioactivity of replicate blots carried out with a Betagen blot
analyzer.
FPGS Gene Copy Number in Parental and Metoprine-resistant
L1210 Cells
Increased levels of FPGS mRNA in the
metoprine-resistant variants (L1210/R83 and L1210/R84 cells) compared
with parental L1210 cells might also be accounted for by an increase in
gene copy number(45) . This possibility was evaluated by
quantitative dot-blotting and Southern blotting (38) of
restriction enzyme digested genomic DNA from these various cell lines
using the murine FPGS cDNA probe. The results (data not shown) revealed
no unique restriction polymorphisms in the DNA and no evidence for
increased FPGS gene copy number in these variants compared with
parental cells.FPGS mRNA Transcription in Variant and Parental L1210
cells
In light of the results above showing no difference among
these variants in either FPGS mRNA stability or FPGS gene copy number,
we examined the rate of FPGS mRNA transcription in parental L1210 cells
and L1210/R84 and L1210/R25 cells by means of a nuclear run-on
assay(44, 45) . Since dihydrofolate reductase activity
was also increased (26) in L1210/R84 cells, we also probed for
increased transcription of its mRNA in L1210/R84 cells as a positive
control. Dihydrofolate reductase activity was not increased in
L1210/R25 cells. The results presented in Fig. 5show that the
rate of FPGS transcript formation when related to controls with
L1210/R84 derived nuclear material (Fig. 5A) was
markedly increased compared to wild-type, while the relative rate of
transcript formation with L1210/R25 derived nuclear material (Fig. 5B) was decreased. The extent of these
differences was quantitated with the Betagen Blot analyzer and found
(data not shown) to be in general agreement with the differences shown
for FPGS activity and mRNA level. That is, transcription of FPGS mRNA
was increased 7-8-fold in L1210/R84 cells and decreased almost
3-fold in L1210/R25 cells compared with parental L1210 cells.
P-Labeled mRNA transcripts obtained with nuclear extracts
of each cell type were used in a RNA/DNA blot with murine FPGS cDNA.
cDNAs for murine dihydrofolate reductase,
- and -actin, and
plasmid-related neomycin resistance were used as controls. A,
blot obtained with mRNA transcripts from L1210 and L1210/R84 cell
nuclear extracts. B, blots obtained with mRNA transcripts from
L1210 and L1210/R25 cell nuclear extracts done under different
conditions. Additional details are provided in the text. The blot shown
is from a typical experiment replicated
twice.
), the natural diastereoisomer of
5-formyltetrahydrofolate; HPLC, high performance liquid chromatography;
EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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