(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 (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.
Cellular folates exist primarily as -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.
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 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.
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 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.
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 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.
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 -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.
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 -actin mRNA. The data in B are from an analysis of
radioactivity of replicate blots carried out with a Betagen blot
analyzer.
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. 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.
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].