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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 5 (March 1), 1999:
pp. 1677-1683
Role of Folylpolyglutamate Synthetase and Folylpolyglutamate
Hydrolase in Methotrexate Accumulation and Polyglutamylation in
Childhood Leukemia
By
Marianne G. Rots,
Rob Pieters,
Godefridus J. Peters,
Paul Noordhuis,
Christina H. van Zantwijk,
Gertjan J.L. Kaspers,
Karel Hählen,
Ursula Creutzig,
Anjo J.P. Veerman, and
Gerrit Jansen
From the Departments of Pediatric Hematology/Oncology and of Medical
Oncology, University Hospital Vrije Universiteit, Amsterdam; the
Department of Pediatric Hematology/Oncology, Sophia Children's
Hospital, Erasmus University, Rotterdam, the Netherlands; and the
AML-BFM Study Group, Germany.
 |
ABSTRACT |
Inefficient polyglutamylation is a mechanism of resistance to
methotrexate (MTX) in childhood T-lineage acute lymphoblastic leukemia
(T-ALL) and in acute myeloid leukemia (AML) in comparison with
childhood c/preB-ALL. We analyzed the profile of MTX polyglutamylation in childhood c/preB-ALL, T-ALL, and AML (n = 45, 15, and 14, respectively), the activity of the MTX-polyglutamate synthesizing
enzyme folylpolyglutamate synthetase (FPGS) (n = 39, 11, and 19, respectively) and of the MTX-polyglutamate breakdown enzyme
folylpolyglutamate hydrolase (FPGH) (n = 98, 25, and 34, respectively). MTX-Glu4-6 accumulation after 24 hours
exposure to 1 µmol/L [3H]-MTX in vitro was lower in
T-ALL (threefold) and AML (fourfold) compared with c/preB-ALL
(P  .001). The FPGS activity was twofold lower in T-ALL
and AML than in c/preB-ALL samples (P < .01). FPGH activity
was not different between c/preB-ALL and T-ALL, but threefold higher in
AML (P < .001). FPGS, FPGH, and the ratio FPGS/FPGH were
correlated with MTX-Glu4-6 accumulation
(r = .49, r =  .34 and r = .61,
respectively). Multivariate analysis showed that FPGS, but not FPGH,
was an independent contributor for MTX-Glu1-6 accumulation,
but not for MTX-Glu4-6 accumulation. In conclusion, low
FPGS activity is associated with low accumulation of
MTX-Glu4-6 in T-ALL and AML. For the group of AML as
compared with the group of ALL, a high FPGH activity can play an
additional role.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
NOWADAYS, CHILDHOOD acute lymphoblastic
leukemia (ALL) has an event-free survival of 70%, which is in striking
contrast to that for childhood acute myeloid leukemia (AML), which is
approximately 40%.1 Methotrexate (MTX) is an important
drug in the treatment of childhood ALL,2 but clinical
trials have shown that AML patients have low response rates to MTX,
similar to the response rates of clinically resistant relapsed ALL
patients (summarized by Bender3). Although in these trials,
the dosages of MTX administered were low and the number of patients was
limited, MTX is not included in standard therapy protocols for AML.
Little is known about the mechanisms of MTX resistance in pediatric
AML. Studies in a limited number of samples from mainly adult AML
patients have suggested that intrinsic MTX resistance can be ascribed
to lower polyglutamylation capacities of AML cells compared with ALL
cells.4-7 Inefficient polyglutamylation will result in a
decrease of MTX-polyglutamates, especially MTX-Glu4-6, which are preferentially retained intracellularly and provoke inhibition of thymidylate synthase and enzymes involved in purine nucleotide synthesis.8
For childhood ALL, accumulation of MTX and MTX-polyglutamates
was correlated with event-free survival9 and with
short-term antileukemic effect.10 Less efficient
polyglutamylation of MTX was observed in leukemic blasts from children
with T-ALL compared with c/preB-ALL, both in vitro11
and in vivo.12,13
The polyglutamylation defect in T-ALL and AML cells was associated with
a lower activity of folylpolyglutamate synthetase (FPGS), the enzyme
that catalyzes the polyglutamate chain formation, as compared with
c/preB-ALL cells.14 The difference in FPGS activity was not
associated with a decreased FPGS mRNA expression,15 but
with a lower affinity of FPGS for MTX in AML cells.16
Besides decreased synthesis of the glutamate side chain,
polyglutamylation defects may also be caused by increased breakdown of
polyglutamates by folylpolyglutamate hydrolase (FPGH).17 Recently, a number of reports have demonstrated a possible role for
FPGH in contributing to MTX resistance in experimental model systems.
In human soft tissue sarcoma cell lines, intrinsic MTX resistance
resulting from impaired polyglutamylation18 could be
explained by a higher FPGH activity compared with MTX responsive cell
lines.19 H35 rat hepatoma and human CCRF-CEM
leukemia cell lines have recently been reported to acquire MTX
resistance by increasing FPGH activity compared with the MTX
sensitive parental cell lines.20,21
A role for FPGH in clinical resistance to MTX has not been established,
although in a recent report including eight ALL and seven AML samples,
Longo et al.22 reported that the ratio FPGS/FPGH was better
at predicting the amount of MTX-polyglutamates accumulated to determine
either activity alone. In the present study, we examined the FPGS and
FPGH activities as well as the polyglutamylation profile in childhood
leukemia samples and found evidence that inefficient polyglutamylation
was associated with a high FPGH activity in AML, but not in T-ALL.
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MATERIALS AND METHODS |
Patient specimens.
Bone marrow and/or peripheral blood was obtained with informed
consent from 108 children with newly diagnosed common or preB-ALL (c/preB-ALL), 29 children with T-ALL, and 29 children with AML at first
diagnosis before start of therapy. Samples from another six AML
patients were obtained at time of relapse; these patients had not
received MTX previously. Boys represented 56% of the c/preB-ALL patients and 79% of the children with T-ALL. Median age was 51.5 months (range, 15 to 190 months) for c/preB-ALL and 90.5 months for
T-ALL (range, 13 to 179 months). Characteristics of the AML patients
are presented in Table 1.
Mononuclear cells were isolated by Ficoll density gradient
centrifugation as described previously.23 Samples with a
leukemic cell percentage below 80% were enriched for blasts by
removing nonmalignant cells using monoclonal antibodies (MoAbs) linked to magnetic beads (Dynabeads M-450; Dynal Inc, Oslo, Norway) as previously described.24 The samples were washed twice with
RPMI containing 2% fetal calf serum (FCS) and resuspended in culture medium consisting of RPMI 1640 (Dutch modification; GIBCO, Uxbridge, UK) plus 20% FCS, 2 mmol/L L-glutamine, 100 IU/mL penicillin, 100 µg/mL streptomycin, 0.125 µl/mL fungizone, 200 µg/mL gentamycin (all obtained from Flow Laboratories, Irvine, UK), 5 µg/mL insulin, 5 µg/mL transferrin, and 5 ng/mL sodium selenite (purchased from Sigma,
Zwijndrecht, the Netherlands).
Reagents.
Methotrexate was a gift from Pharmachemie (Haarlem, The Netherlands).
MTX-Glu2 was purchased from Schircks Company (Jona, Switzerland). [3,5,7-3H]-MTX (20 Ci/mmol) was obtained
from Moravek Biochemicals (Brea, CA). [2,3-3H]-L-glutamic
acid (19 Ci/mmol) formulated in 0.01 N HCl (NET 395) was provided by
New England Nuclear (Boston, MA). Other reagents used were of
analytical grade.
MTX polyglutamylation.
Ten million freshly isolated leukemic cells were incubated for 24 hours
with 1 µmol/L 3H-MTX (final specific activity 2 Ci/mmol)
in 5 mL culture medium. After the cells were harvested and washed three
times in phosphate-buffered saline (PBS) by centrifugation (5 minutes
at 300g, 4°C), the pellet was resuspended in 1 mL PBS. A
sample of 90 µL was counted for radioactivity, 10 µL was used to
determine the number of cells (including trypan blue positive cells);
the remaining suspension was centrifuged 5 minutes at 12,000g
and the pellet was kept at 20°C until extraction.
To extract the polyglutamates, the pellet was resuspended in 150 µL
ice-cold PBS and left on ice for 20 minutes after the addition of 50 µL 40% trichloroacetic acid (TCA). After centrifugation, 400 µL
Tri-octylamine/1,1,2-tri-chloro-tri-fluoro-ethane (1/4, vol/vol) was
added to neutralize the extract. After vortexing and centrifugation,
the upper waterlayer was stored at 20°C until HPLC analysis.
MTX-polyglutamates were analyzed using an anion exchange column
(Partisphere SAX, Whatman, I.D. 4.6 mm, length 12.5 cm, particle size 5 µm) running 4 minutes with 98% buffer A (60 mmol/L
NH4H2PO4, pH 5.5) and 2% buffer B
(600 mmol/L NH4H2PO4, pH 5.5),
followed by a gradient for 16 minutes to 100% buffer B.25
This was subsequently reduced to 2% buffer B over 5 minutes and
maintained for another 5 minutes. UV detection was at 309 nm. The data
are expressed as pmol MTX-Glun/109
cells, which allows comparison with previously described
values.7,9,10,12,13
Folylpolyglutamate synthetase.
FPGS activity was assayed as described in detail by Jansen et
al.26 Briefly, 15 × 106 freshly isolated
cells were washed twice in PBS and suspended in 250 µL extraction
buffer (50 mmol/L Tris-HCl, 20 mmol/L KCl, 10 mmol/L MgCl2,
and 5 mmol/L dithiotreitol (DTT), pH 7.6). Crude cell extracts were
obtained by sonification (three times for 5 seconds at 14 micron, with
10 seconds intervals, at 4°C) followed by centrifugation (15 minutes,
12,000g, at 4°C). The protein content of the supernatant was
determined using the Biorad protein assay.27 The FPGS assay
mixture contained in a volume of 250 µL: 200 µg protein, 4 mmol/L
[3H]-L-glutamic acid ([3H]-Glu; final
specific activity: 6.6 Ci/mol) and 250 µmol/L MTX in 100 mmol/L Tris,
10 mmol/L ATP, 20 mmol/L MgCl2, 20 mmol/L KCl, and 10 mmol/L DTT at a pH of 8.85. After 2 hours incubation at 37°C, during
which the reaction was proven to be linear, the reaction was stopped by
the addition of 1 mL 5 mmol/L ice-cold, nonlabeled L-glutamic acid.
MTX-[3H]-Glu2 was separated from unreacted
[3H]-glutamic acid by Sep-Pack C18 reverse
phase column chromatography (Millipore, Waters Associates, Etten-Leur,
The Netherlands). The amount of MTX-3[H]-Glu2
was measured by direct  -scintillation counting. Controls without MTX
were subtracted to correct for polyglutamylation of endogenous folates
present in the samples. HPLC analysis demonstrated that under these
conditions, MTX-Glu2 was the only product formed. The FPGS
activity is expressed as pmol MTX-Glu2 formed per hour per
106 cells for comparison with polyglutamylation parameters,
which are expressed per 109 cells. FPGS activities
expressed per milligram of cellular protein are also provided.
Viability of the samples before protein extraction was not of influence
on the reported FPGS activities (r = .06, P = .7, n = 62).
Folylpolyglutamate hydrolase.
The FPGH activity was assayed as described by O'Connor et
al.28 Briefly, 2 × 106 cells were washed
twice in PBS and suspended in 100 µL extraction buffer (100 mmol/L
Tris-HCl, pH 6.9). Crude cell extracts were obtained by sonification
(three times for 5 seconds at 14 micron, with 10 seconds intervals, at
4°C) followed by centrifugation (15 minutes, 12,000g, at
4°C). The protein content of the supernatant was determined using the
Biorad protein assay.27 The FPGH reaction mixture contained
25 µg protein and 20 nmol MTX-Glu2 in 200 µL 100 mmol/L
Tris-HCl, pH 6.9 and was incubated for 1 hour in a 37°C waterbath.
Under the conditions used, the reaction was linear. The reaction was
stopped by heating the samples for 3 minutes at 95°C. After cooling
on ice for 15 minutes, the samples were centrifuged (15 minutes,
12,000g, 4°C) and the supernatant was stored at 20°C
until HPLC analysis. MTX-Glu2 was separated from the
product MTX (ie, MTX-Glu1) by HPLC as described for the
polyglutamate chain length analysis. Both freshly obtained and
cryopreserved samples were analyzed, since cryopreservation had no
effect on FPGH activity as observed by us and by others.29
FPGH activity is expressed as nanomoles MTX formed per hour per
milligram of protein or as nanomoles MTX formed per hour per
106 cells for comparison with other MTX polyglutamylation
parameters. Viability of the samples before protein extraction was not
of influence on the reported FPGH activities (r = .09,
P = .3, n = 106).
Statistical analysis.
The Mann-Whitney U-test was used to compare c/preB-ALL with T-ALL and
AML data. To determine any relation of the MTX-polyglutamylation parameters, the Spearman correlation test was applied. Multivariate statistical comparisons were conducted including phenotype, FPGS, FPGH,
and accumulation of MTX-Glu1-6 or accumulation of
MTX-Glu4-6. Analyses were two-tailed at the significance
level of P < .05.
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RESULTS |
MTX accumulation and polyglutamylation.
Total MTX accumulation, ie, MTX-Glu1-6, in samples from
c/preB-ALL patients (n = 45) ranged from 205 to 4,838 pmol/109 cells as shown in Fig
1. The range was more narrow for 15 T-ALL and 14 AML samples tested (maximum accumulation, 1,943 and 2,103 pmol
MTX-Glu1-6/109 cells, respectively); no T-ALL
or AML sample accumulated more total MTX than the 75th percentile of
the c/preB-ALL samples. The median amount of total MTX accumulation was
1.5-fold lower in T-ALL compared with c/preB-ALL samples (888 v
1,321 pmol/109 cells, respectively; P = .02), but
was not significantly different between AML and c/preB-ALL samples
(1,216 v 1,321 pmol/109 cells, respectively).
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| Fig 1.
Accumulation of total MTX, ie, MTX-Glu1-6
(pmol/109 cells; left panel) and of long chain
polyglutamates, ie, MTX-Glu4-6 (pmol/109 cells;
right panel) in c/preB-ALL (n = 45), T-ALL (n = 15), and AML
cells (n = 14) after 24 hours in vitro incubation with 1 µmol/L
[3H]-MTX. Each patient sample is represented by a dot,
lines represent the median values.
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Separation of the accumulated MTX-polyglutamates based on chain length
resulted in different patterns for c/preB-ALL, T-ALL, and AML. A
spectrum with increasing amounts from unmetabolized MTX to
MTX-Glu5 was found for c/preB-ALL samples as shown in Fig 2. For T-ALL, similar amounts of MTX-Glu1 to
MTX-Glu5 were observed, whereas for AML samples the main
metabolites were MTX-Glu1-3 (Table 1).
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| Fig 2.
Distribution of MTX metabolites (MTX-Glu1-6)
expressed as mean value pmol
MTX-Glun/109 cells (±SE) for 40 c/preB-ALL ( ), 13 T-ALL ( ), and 14 AML ( ) patients. Leukemic
cells were incubated for 24 hours with 1 µmol/L
[3H]-MTX as described in Materials and Methods.
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The median percentage of MTX present as the pharmacologically more
important long-chain polyglutamates MTX-Glu4-6 was 66% for
the c/preB-ALL cells (range, 34% to 90%) compared with 42% in the
T-ALL samples (range, 19% to 85%; P < .001) and 29% in the AML samples (range, 0% to 62%; P < .001).
Consequently, the median absolute amount of MTX-Glu4-6 was
906 pmol/109 cells in c/preB-ALL samples versus 290 pmol/109 T-ALL cells (P = .001) and 225 pmol/109 AML cells (P < .001) (Fig 1).
FPGS and FPGH activity.
Samples of 39 children with c/preB-ALL, 11 with T-ALL, and 19 with AML
contained sufficient cells to be assayed for FPGS activity (Table
2). The median FPGS activity for c/preB-ALL samples was twofold higher than the median FPGS activity for T-ALL or AML samples
(P < .01). When the activity was normalized per milligram of cellular protein, comparable differences were found.
The FPGH activity of ALL samples showed a broad range as depicted in
Table 2 and Fig 3. The median FPGH activity was not different between 94 c/preB-ALL and 24 T-ALL samples. A narrow range in
FPGH activities was found for the AML cells with the exception of one
outlier in which the FPGH activity was below detection limit. The
median value was threefold higher for the AML samples compared with the
c/preB-ALL samples when expressed per cell (P < .001). The
FPGH activity normalized per milligram of cellular protein was 1.4-fold
higher in AML cells compared with c/preB-ALL cells (P = .05;
Table 2). Differences in FPGH activity between the small groups of AML
subtypes (mainly M1, M2, and M4) were not statistically significant
(Table 1). FPGH activity was not related to FPGS activity in the total
group of 62 acute leukemia samples (r = .09;
P = .5), nor in the separated groups of 36 c/preB-ALL, 9 T-ALL, and 17 AML samples.
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| Fig 3.
FPGH activities in cell extracts of 94 c/preB-ALL, 24 T-ALL, and 33 AML samples determined by incubation with 100 µmol/L
MTX-Glu2 as a substrate for FPGH. Data are expressed as
nmol MTX formed/h/106 cells.
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Correlation of FPGS or FPGH with polyglutamylation.
A significant correlation was found between FPGS activity and
accumulation of MTX-Glu1-6 in 20 c/preB-ALL samples
analyzed for these parameters (r = .62; P = .004).
The same was true for FPGS activity and accumulation of
MTX-Glu4-6 (r = .49; P = .04) (Table
3). Inclusion of these parameters from eight T-ALL and nine AML samples, resulted in similar findings, as shown in Fig 4A
for 33 samples of which FPGS and MTX-Glu4-6 accumulation
data were available.
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Table 3.
Correlations of FPGS, FPGH, and the Ratio FPGS/FPGH with
MTX Accumulation and Polyglutamylation Data in Childhood Leukemia
Samples
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| Fig 4.
(A) Correlation of FPGS activity (pmol
MTX-Glu2 formed/h/106 cells) with accumulation
of MTX-Glu4-6 (expressed as pmol
MTX-Glu4-6/109 cells). (B) Correlation of FPGH
activity (nmol MTX formed/h/106 cells) with accumulation of
MTX-Glu4-6 (expressed as pmol
MTX-Glu4-6/109 cells). (C) Correlation of the
ratio FPGS/FPGH activities expressed per 106 cells with
accumulation of MTX-Glu4-6 (expressed as pmol
MTX-Glu1-6/109 cells). Spearman rank
correlation coefficients are presented for the total group. Statistical
parameters for c/preB-ALL, T-ALL, and AML samples are presented
separately in Table 3. ( ), c/preB-ALL; ( ), T-ALL; (), AML.
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No correlation between FPGH and accumulation of MTX-Glu1-6
was observed in the separate subgroups or in the total group of 61 acute leukemia samples. The importance of FPGH activity for the
differences in MTX polyglutamylation between T-ALL, AML, and c/preB-ALL, however, was suggested by the significantly inverse relation between FPGH and the accumulation of MTX-Glu4-6
(r = .34, P = .01) (Fig 4B). No correlation was
observed between FPGH activity and the accumulation of
MTX-Glu4-6 when analyzed within the separate groups of ALL
or AML samples (Table 3).
For 29 patients, FPGS and FPGH activities as well as
MTX-Glu4-6 accumulation data were available. In this group
a strong and significant positive correlation was observed for the
FPGS/FPGH ratio with the total MTX accumulation (r = .63;
P < .001) and with the accumulation of
MTX-Glu4-6 (r = .61; P < .001) as
shown in Fig 4C. The correlation of the FPGS/FPGH ratio with
accumulation of MTX-Glu4-6 was stronger than that of the
FPGS or FPGH activities independently with accumulation of
MTX-Glu4-6 in this group of samples (r = .41;
P = .03 and r = .46; P = .01,
respectively). Multivariate regression analysis with inclusion of FPGS,
FPGH, and phenotype, showed that FPGS was the independent predictive factor for the accumulation of MTX-Glu1-6, but not for the
accumulation of MTX-Glu4-6. FPGH did not independently
contribute to polyglutamylation.
| |
DISCUSSION |
In this study, we show that inefficient polyglutamylation, a potential
mechanism for intrinsic MTX resistance in AML cells, can be explained
by a lower FPGS activity along with a higher FPGH activity in AML cells
compared with c/preB-ALL cells. Also for T-ALL, inefficient
polyglutamylation was associated with a lower FPGS activity, but not
with a higher FPGH activity. Long-chain polyglutamates are known to be
preferentially retained intracellularly.8 Because relative
resistance to MTX observed in T-ALL and AML cells compared with
c/preB-ALL was associated with a rapid efflux of the
drug,4,30 defects in polyglutamylation provide a plausible explanation for this phenomenon. Compared with ALL, shorter chain MTX-polyglutamates have actually been observed in adult AML
samples6,7 and in four pediatric AML samples.5
Also the lower response rate to MTX in T-ALL might at least partially
be explained by defects in polyglutamylation.11-13
In the present study consisting of a large group of pediatric leukemia
samples, the distribution pattern of the MTX polyglutamates was
different in c/preB-ALL versus T-ALL and AML. Consistent with other
studies,12,31 we observed that in c/preB-ALL, MTX
was metabolized to long-chain polyglutamates with MTX-Glu5
as the main metabolite. For AML, however, MTX-Glu1-3 were
observed as the major metabolites. The accumulation of the
pharmacologically more important MTX-Glu4-6 was lower in
T-ALL (threefold) and AML (fourfold) compared with c/preB-ALL cells,
which may contribute to the differential MTX sensitivity of these leukemias.
Inefficient MTX polyglutamylation in T-ALL and AML cells has previously
been explained by a lower FPGS activity compared with c/preB-ALL
cells.14 In the present study, the same differences between
c/preB-ALL, T-ALL, and AML were observed. Since polyglutamylation is a
dynamic process of synthesis and breakdown, not only the FPGS activity
but also the activity of FPGH, which hydrolyzes the polyglutamates,
should be taken into account. Laboratory studies using cell lines have
shown that increased FPGH activity conferred a shift in the
distribution of the folylpolyglutamates towards the short-chain
polyglutamates.32 In addition, MTX resistance has been
associated with a high FPGH activity in several human sarcoma cell
lines, in a human leukemia cell line and in a rat hepatoma cell
line.19-21 Moreover, incubation of leukemic blasts with
2-mercapto-methylglutaric acid (MMGA), an inhibitor of FPGH, resulted
in a polyglutamylation pattern for AML cells resembling the pattern
observed for c/preB-ALL samples.33
We investigated the activity of FPGH in 151 pediatric acute leukemia
samples and found no difference between c/preB-ALL, and T-ALL, but a significant three-fold higher FPGH activity in AML cells
compared with c/preB-ALL cells. Since the accumulation of total MTX per
106 cells was similar for AML and c/preB-ALL cells,
relatively more FPGH enzyme per picomole accumulated MTX is present in
AML cells compared with c/preB-ALL cells. This might partially explain
the lower amounts of MTX-Glu4-6 observed in AML compared
with c/preB-ALL. Although AML cells are larger than ALL cells, a
difference in FPGH activity was also observed when the activity was
expressed per milligram of cellular protein. This suggests that a high
FPGH activity contributes to intrinsic MTX resistance in childhood AML.
Given the intracellular compartmentation of FPGH, extrapolation of the
FPGH activity measured in cell extracts to the in vivo situation,
however, should be made cautiously. Studies on tumor cell
lines32,34 have shown that FPGH is primarily localized in
the lysosomes and therefore lysosomal transport of the polyglutamates is likely to be a limiting factor in the breakdown of
MTX-polyglutamates by FPGH.35 In addition, secretion of the
enzyme has been observed in tumor cell lines, which could imply that
FPGH activity as measured in cell free extracts is an overestimation of
the intracellular functional activity.20,28,29
A number of reports demonstrated that the affinity of FPGH for MTX
metabolites increased with increasing polyglutamate side chain
length.35,36 Therefore, differences in FPGH activity between AML and ALL cells, as reported in this study, might be more
pronounced when assayed using MTX-Glu5 as a substrate.
However, we37 and others18,36 observed that
human FPGH displays an exopeptidase activity, ie, sequentially
hydrolyzing the outermost glutamate residue. This results in a complex
cleavage pattern with hydrolyzed products subsequently serving as
substrates again. For a more straightforward determination of FPGH
activity, we used MTX-Glu2 as a substrate, which will be
hydrolyzed to MTX.
To investigate the relative contribution of FPGS and FPGH in the
process of polyglutamylation, we determined the relation of these
enzymes to in situ MTX polyglutamylation irrespectively of the type of
leukemia. A significant strong correlation for both FPGS and FPGH
activity with the accumulation of MTX-Glu4-6 was found.
This relation was stronger when, within the same group of samples, the
ratio of FPGS/FPGH was used instead of single enzyme activity,
indicating that the relative contribution of both enzymes is of
importance as suggested recently in a study with 15 childhood acute
leukemia samples.22 No correlation could be observed for
FPGH and accumulation of MTX-Glu4-6 within the (sometimes small) separate subgroups. However, for the total population of leukemic samples, a role for FPGH in the process of
polyglutamylation seems evident as illustrated by Fig 4A-C.
Multivariate analysis, including FPGS, FPGH, and phenotype, did not
demonstrate that either enzyme activity contributed independently to
the accumulation of MTX-Glu4-6. However, this study shows
that, compared with c/preB-ALL, FPGH is significantly higher in AML while the accumulation of MTX-Glu4-6 is significantly lower
in AML. So, for the group of AML compared with the group of ALL, FPGH
might be a significant factor while the differences in FPGH activity
within one subtype of leukemia do not contribute to polyglutamylation defects. The absence of a causal relation for FPGH on
MTX-Glu4-6 might be explained by additional factors
contributing to the accumulation of MTX-Glu4-6. These
factors may include diminished MTX transport and high levels of DHFR,
which may reduce the pool of MTX available for
polyglutamylation.38
Interestingly, a multivariate model to predict accumulation of
MTX-Glu1-6 showed FPGS as the only independent predictor.
Since MTX accumulation has been described to be a prognostic factor within a group of c/preB-ALL patients,9 this finding might provide a molecular tool to predict MTX-based therapy outcome. In this
respect, mRNA expression levels of FPGS, as reported to be correlated
to functional FPGS activity,13,15 are currently being
investigated in our laboratory.39
This report focuses on polyglutamylation defects as a resistance
mechanism to MTX, but other factors can also contribute to MTX
resistance including (1) defects at the level of membrane transport as
has been reported to occur in one third of AML samples,40 (2) increased levels of the main target enzyme DHFR, (3) mutations in
the DHFR gene leading to decreased affinity for MTX, or (4) presence of
functionally active efflux systems (reviewed by Bertino2 and Peters and Jansen41). Although the first two mechanisms have been described as playing a role in MTX resistance in childhood ALL (reviewed by Pieters et al,42 Jansen and
Pieters,43 Gorlick et al44), polyglutamylation
defects seem to be more predominant factors underlying the presumed
clinical MTX resistance described for AML cells.5
In conclusion, an important independent role is described for FPGS in
predicting the overall MTX accumulation in childhood leukemia subtypes.
A high FPGH activity is associated with a low accumulation of
MTX-Glu4-6 in childhood AML, but not in T-ALL. These data
suggest that inhibitors of FPGH29 or novel antifolates for
which FPGH has a decreased affinity compared with MTX might offer new
approaches to circumvent MTX resistance and to treat children with AML.
| |
ACKNOWLEDGMENT |
The authors thank Dr P.D. Bezemer of the Department of Epidemiology and
Biostatistics, Vrije Universiteit, Amsterdam, the Netherlands for
helpful discussions during the preparation of this manuscript.
| |
FOOTNOTES |
Submitted January 22, 1998; accepted October 22, 1998.
Supported by Grant No. VU 94-679 from the Dutch Cancer Society.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Marianne G. Rots,
Department of Pediatric Hematology/Oncology, University Hospital Vrije
Universiteit, PO Box 7057, 1007 MB Amsterdam, the
Netherlands; e-mail: marianne.rots{at}azvu.nl.
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Abstract
Introduction