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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 1067-1074
Differential Methotrexate Resistance in Childhood T- Versus
Common/PreB-Acute Lymphoblastic Leukemia Can Be Measured by an In Situ
Thymidylate Synthase Inhibition Assay, But Not by the MTT Assay
By
Marianne G. Rots,
Rob Pieters,
Gert-Jan L. Kaspers,
Christina H. van Zantwijk,
Paul Noordhuis,
Rob Mauritz,
Anjo J.P. Veerman,
Gerrit Jansen, and
Godefridus J. Peters
From the Departments of Pediatric Hematology/Oncology and Medical
Oncology, University Hospital Vrije Universiteit, Amsterdam, The
Netherlands.
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ABSTRACT |
Methotrexate (MTX) is not cytotoxic to patient-derived acute
lymphoblastic leukemia (ALL) cells in total-cell-kill assays, such as
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay, putatively due to the rescue effects of hypoxanthine and
thymidine released from dying cells. This was mimicked by a diminished
methotrexate (MTX) cytotoxicity for the cell lines HL60 and U937 in the
presence of hypoxanthine, thymidine, or lysed ALL cells. However,
enzymatic depletion or inhibition of nucleoside membrane transport did
not result in MTX dose-dependent cytotoxicity in patient samples.
Alternatively, a thymidylate synthase inhibition assay (TSIA), based on
inhibition of the TS-catalyzed conversion of 3H-dUMP to
dTMP and 3H2O, correlated with the
MTT assay for antifolate sensitivity in four human leukemia cell lines
with different modes of MTX resistance. For 86 ALL patient samples,
TSI50 values after 21 hours exposure to MTX were not
different between T- and c/preB-ALL (P = .46). After 3 hours
incubation with MTX followed by an 18-hour drug-free period, T-ALL
samples were 3.4-fold more resistant to MTX compared with c/preB-ALL
samples (P = .001) reflecting the clinical differences in MTX
sensitivity. TSI50 values correlated with MTX accumulation
(r =  .58, P < .001). In conclusion,
the TSIA, but not the MTT assay, can measure dose-response curves for
MTX in patient-derived ALL cells and showed relative MTX resistance in
T-ALL compared with c/preB-ALL.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ACUTE LYMPHOBLASTIC leukemia (ALL) is the
most frequently occurring type of cancer in children. Today, more than
95% of the children with newly diagnosed ALL will reach complete
remission by combination chemotherapy. However, the leukemia will
relapse in one third of the patients.1 This treatment
failure may be explained by unfavorable clinical
pharmacokinetics,2 regrowth potential of the residual
leukemic cells, and by cellular drug resistance.3 Testing
of in vitro drug resistance, eg, by using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay, has provided good correlations with clinical
outcome.4-8 Unfortunately, the cytotoxicity of methotrexate
(MTX), a cornerstone in the treatment of ALL, cannot be evaluated on
primary patient-derived leukemic cells by the MTT assay9-12
or by other 4-day culture assays, such as the differential staining
cytotoxicity (DiSC) assay.13 A test system, which allows
the determination of MTX resistance of primary ALL cells, might provide
additional prognostic information, thereby facilitating the
identification of low- and high-risk patients. In addition, an in vitro
assay may be used to determine the relation between clinical and cell
biological features and MTX resistance.
In contrast to the nonproliferating patient-derived ALL cells,
MTX-induced growth inhibition can easily be observed in cell lines
using the MTT assay.14 The mechanism of action of MTX is
through the inhibition of folate requiring enzymes in the pyrimidine and purine de novo synthesis pathways, ultimately resulting in an
inhibition of the synthesis of DNA, RNA, and protein.15
However, cells can be rescued from this cytotoxic action by salvage
pathways in which, eg, hypoxanthine (Hx) is converted to inosine
monophosphate (IMP) and subsequently to adenosine
monophosphate (AMP) and guanosine monophosphate (GMP).
Inhibition of the de novo synthesis of thymidylate catalyzed by
thymidylate synthase (TS) can be bypassed by the consumption of
exogenous thymidine (TdR), which will be converted by thymidine kinases
to dTMP. Hx and TdR have been reported to be released by spontaneously
dying lymphocytes16 due to degradation of DNA and RNA.
Because on average 35% of ALL cells derived from patients
spontaneously die during 4 days of culture,10 thereby releasing Hx and TdR, this may explain the absence of a dose-response curve of MTX for ALL cells. Additional evidence for this hypothesis is
provided by a report in which enzymatic depletion of Hx and TdR
resulted in MTX cytotoxicity on GKTL cells, a leukemia xenograft, which
is unable to grow ex vivo, similar to ALL cells.17 This approach, however, was not applied to samples directly derived from
patients, and so far it is unknown whether total-cell-kill assays can
be adapted for the screening of cells derived from pediatric ALL
patients for MTX sensitivity.
In the present study, we investigated several adaptations of the MTT
assay for the screening of ALL cells from childhood leukemia patients
for MTX sensitivity or resistance, but no dose-dependent cytotoxicity
of MTX was observed. Alternatively, we showed that an in situ
thymidylate synthase inhibition assay (TSIA)18,19 could be
used to determine relative sensitivities of cell lines to MTX,
obtaining similar data as for the MTT assay. Therefore, we optimized
this indirect MTX resistance assay for the screening of patient-derived
leukemic cells. The results show that the TSIA provides dose-response
curves in patient-derived ALL cells with large interpatient variation
and that this assay can detect differential MTX resistance in T-
versus c/preB-ALL after short-term exposure. This is in accordance
with the clinically observed relative chemoresistance of T-ALL compared
with c/preB-ALL cells.20
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MATERIALS AND METHODS |
Patient specimens.
Bone marrow and peripheral blood samples were obtained from newly
diagnosed pediatric ALL patients; infants (<12 months old) and
patients with proB- (CD10 precursor B-lineage) and
mature B-ALL were excluded. Mononuclear cells were isolated by
centrifugation (500g, 25 minutes) with Ficoll Isopaque, as
described previously.9 After isolation, cells were washed
twice in RPMI containing 1% fetal calf serum (FCS) with 10-minute
periods of centrifugation at 300g and suspended at 2 × 106 cells/mL in culture medium (RPMI 1640 containing 20%
heat-inactivated FCS, 100 IU/mL penicillin, 100 µg/mL streptomycin,
0.125 µg/mL fungizone, 200 µg/mL gentamycin, 2 mmol/L L-glutamine,
5 µg/mL insulin, 5 µg/mL transferrin, and 5 ng/mL sodium selenite).
Remaining cells were cryopreserved in RPMI containing 20% FCS and 10%
dimethyl sulfoxide (DMSO) in liquid nitrogen.
Cell lines.
HL60, a human promyelocytic leukemia cell line, and U937, derived from
a patient with monoblastic leukemia, and four human T-lymphoblastic
leukemia cell lines CCRF-CEM (the parental CEM/S and three
MTX-resistant sublines with either defective MTX transport, increased
dihydrofolate reductase [DHFR], or defective
MTX-polyglutamylation)21 were grown as suspension cultures
in RPMI medium 1640 supplemented with 10% heat-inactivated FCS and 1 mmol/L L-glutamine. Cultures were maintained in exponential growth at
37°C in a humified atmosphere of 95% air/5% carbon dioxide. In
the experimental set up, HL60 cells were suspended at 0.2 × 106 cells/mL, U937 and CEM cells at 0.1 × 106 cells/mL for the MTT assay and at 2 × 106 cells/mL for the TSIA.
Chemicals.
MTX was obtained as a gift from Pharmachemie (Haarlem, The
Netherlands). Fluoro-MTX (F-MTX), a nonglutamylatable MTX
analogue,22 was kindly provided by Dr J.J. McGuire (Grace
Cancer Center, Buffalo, NY). AG337, edatrexate, GW1843U89,
trimetrexate, and ZD1694 were provided by Agouron Pharmaceuticals (La
Jolla, CA), Ciba-Geigy (Basel, Switzerland), Glaxo Wellcome (Research
Triangle Park, NC), Werner-Lambert/Parke Davis (Ann Arbor, MI), and
Zeneca (Maccles Field, UK), respectively. FCS, penicillin,
streptomycin, fungizone, gentamycin, and L-glutamine were obtained from
Flow Laboratories (Irvine, UK), bovine serum albumin from Organon
Technika (Oss, The Netherlands), and Ficoll Isopaque (Lymphoprep 1.077 g/mL) was provided by Nyegaard (Oslo, Norway). Insulin, transferrin, sodium selenite, and MTT were purchased from Sigma (Zwijndrecht, The
Netherlands), as well as thymidine, hypoxanthine, thymidine phosphorylase (TP), and xanthine oxidase (XO).
2-(aminocarbonyl)-N-(4-amino-2,6-dichlorophenyl)-4-[5,5-bis-(4-fluorophenyl-l0-phenyl]-1-piperazineacetamide (R75231) was kindly provided by Dr H. van Belle (Jansen
Pharmaceuticals, Beerse, Belgium).
[5-3H]-2'-deoxyuridine (22 Ci/mmol) and
[5-3H]-2'-deoxycytidine (25 Ci/mmol) were purchased
from Moravek Biochemicals (Brea, CA).
MTT and DiSC assay in blast cells from patients.
Round-bottomed 96-well microculture plates (Costar, Badhoevedorp, The
Netherlands) were filled with 20 µL of different MTX concentrations
and stored at 20°C. Aliquots of 80 µL of the cell suspension (2 × 106 cells/mL) were added to each well
as described previously.9 Four wells contained medium
without drugs or cells for blanking the plate reader and eight wells
contained cells and no drug for measuring control cell viability.
Twelve concentrations of MTX ranging from 51.2 pg/mL to 2,500 µg/mL
(0.11 nmol/L to 5.5 mmol/L) were tested in duplicate. Plates were
incubated in a humified incubator in 5% CO2 for 4 days at
37°C.
On day 4, 10 µL of MTT solution (final concentration, 0.45 mg/mL) was
added and after shaking until the cell pellet was resuspended, the
plate was incubated at 37°C for 6 hours. Formazan crystals were
dissolved with 100 µL of 0.04 N HCl-isopropyl alcohol (acid isopropanol). The optical density (OD) of the wells was measured using
an EL312 microplate spectrophotometer (Biotek Instruments Inc,
Winooski, VT) at 540 and 562 nm. The OD is linearly
related to cell number.23 The OD of wells containing MTX,
but no cells, was subtracted from the OD of corresponding wells
containing cells and MTX. Leukemic cell survival (LCS) was calculated
by the equation: LCS = (ODday 4 treated wells/ODday
4 control wells) × 100%. In simultaneous experiments, the
medium was preincubated for 15 minutes with TP (final concentration,
0.15 IU/mL) and XO (final concentration, 0.02 IU/mL) at 37°C to
deplete TdR and Hx. Nucleoside transport was inhibited by the addition
of R75231 (final concentration, 1.65 µmol/L). The DiSC assay was
performed as described elsewhere.24
MTT and DiSC assay in cell lines.
Individual wells of a 96-well microculture plate were filled with 50 µL cell suspension, 25 µL medium, and 25 µL MTX (final concentrations ranging from 1011 to
105 mol/L). Plates were incubated in a humified
incubator in 5% CO2 for 3 days at 37°C. Subsequently,
10 µL of MTT solution (final concentration, 0.45 mg/mL) was added and
incubated for 3 hours at 37°C. Formazan crystals were dissolved
with 100 µL of 0.04 N HCl-isopropyl alcohol (acid isopropanol). The
concentration resulting in 50% inhibition of the cell growth
(IC50) was determined by assigning 100% to the mean value
for the OD of the control wells at day 3 after subtraction of the mean
blank value. To distinguish between cell growth inhibition and cell
kill, the OD after 3 days was corrected for the mean OD observed for
the control wells at the day of drug addition.25 In some
experiments, Hx, TdR, or lysed ALL cells were added to the medium and
IC50 values were determined relative to control cells also
incubated in the presence of these additives. Lysed leukemic cells
(final concentration, 1 × 106/mL) were obtained by
snap-freezing and thawing three times.
Measurements of Hx and TdR.
After 4 days of culture, microculture plates were kept at
20°C, at which temperature Hx and TdR are
stable,26 until analyzed. To determine the concentration of
Hx and TdR, 7 µL of 80% trichloroacidic acid (TCA) was added to 70 µL medium and put on ice for 30 minutes. After 2 minutes of
centrifugation at 12,000g, the supernatant was neutralized by
the addition of 140 µL of
tri-octylamine/1,1,2-tri-chloro-tri-fluoro-ethane (1/4; vol/vol). This
mixture was centrifuged for 1 minute at 12,000g. The separation
of the nucleosides casu quo bases in the supernatant was
performed on a reverse-phase microsphere C18 column (Chrompack; 100 × 4.6 mm, 3 µm) at a flow rate of 1 mL/min. Eluents were made of 60 mmol/L KH2PO4 in 5 mmol/L
tetrabutylammonium hydrogensulphate (TBA), at pH 6 (solvent A) and 50%
acetonitril (solvent B). A gradient (2 minutes 2% B, followed by
linear increase of 2% to 40% B over 10 minutes) separated the peaks,
which were identified by their absorbance ratios and retention times by
comparison with standards. Column output was monitored at 254 and 280 nm.
In situ TSIA.
Inhibition of TS was determined in whole cells based on a previously
described assay18,19 by measuring the TS-catalyzed conversion of 3H-dUMP to dTMP and
3H2O. A total of 135 µL cell suspensions (1 × 106 cells/mL; 4 × 106 cells/mL
when deoxyuridine was used as a substrate) were incubated at 37°C
with either 15 µL RPMI as controls or with 15 µL MTX solution. Blanks in triplicate were included containing 135 µL culture medium and 15 µL RPMI. Two conditions were tested: (1) continuous incubation in which cells were incubated with or without drugs for 21 hours (five
final concentrations ranging from 0.0039 µmol/L to 1 µmol/L for
MTX; from 0.039 µmol/L to 10 µmol/L for F-MTX ) and (2) short exposure in which the drug was washed away after 3 hours followed by an
18-hour drug-free period (five final MTX concentrations ranging from
0.156 µmol/L to 40 µmol/L), based on pilot experiments reported
elsewhere.27 Controls were included in triplicate for both
conditions. [5-3H]-2'-deoxycytidine or
[5-3H]-2'-deoxyuridine (final concentration, 1 µmol/L, 2.5 Ci/mmol) was added 4 hours after the start of the
experiment as precursor for dUMP, the substrate for TS. After a total
incubation time of 21 hours, cells were put on ice and 150 µL 35%
ice-cold TCA was added together with 750 µL 10% activated charcoal
solution (10 g washed charcoal, 0.5 g dextran, and 2.5 g
bovine serum albumin [BSA] in 100 mL demineralized water) as
described previously for the in vitro TS catalytic activity
assay.28 After vortexing, samples were left on ice for 30 minutes and centrifuged (800g, 30 minutes, 4°C); 450 µL
of the aqueous phase, containing 3H2O, was
transferred to a scintillation vial and counted for radioactivity. After subtraction of the mean blank counts, the data were evaluated by
calculation of the concentration of drug needed to inhibit 50% of the
control TS activity, assuming a linear dose-response curve between the
two flanking concentration points. Data were expressed as
TSI50,cont. referring to the continuous exposure condition and as TSI50,short for the short exposure
condition. Experiments in which cell lines were assayed for
antifolate sensitivity were performed by incubating 2 × 106 cells/mL applying the same time schedule. In this case,
3H-deoxyuridine was added as a substrate 1 hour before the
end of the experiment (final concentration, 1 µmol/L, 0.7 Ci/mmol).
MTX accumulation and polyglutamylation.
Of samples for which a sufficient number of cells was available,
107 cells were exposed for 24 hours to 1 µmol/L
[3H]-MTX (2 Ci/mmol), washed three times in ice-cold
phosphate-buffered saline (PBS) and resuspended in 1 mL ice-cold PBS.
Total accumulation was determined by counting 90 µL for radioactivity
and 10 µL for cell survival. The remaining 900 µL was centrifuged
and analyzed for polyglutamate formation by high-performance liquid
chromatography (HPLC) as described previously.29
Statistics.
To analyze correlations between parameters obtained with the TSIA and
with the MTT assay, the Wilcoxon signed rank sum test was applied. The
Spearman's rank correlation coefficient was calculated for a relation
between MTX sensitivity and polyglutamylation parameters. The
Mann-Whitney U test was performed to determine differences in MTX
sensitivity between T- and c/preB-ALL. All tests were performed by
applying SPSS 7.5 for Windows (SPSS Benelux BV, Gorinchem, The
Netherlands) software.
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RESULTS |
Prevention of MTX-induced growth inhibition on leukemic cell lines.
A dose-dependent growth inhibition was induced by MTX for the leukemic
cell lines HL60 and U937, as determined with the MTT assay
(IC50 values, 8.8 nmol/L ± 1.0 and 11.0 nmol/L ± 4.0, respectively). For both cell lines, cell growth was completely
inhibited at MTX concentrations higher than 107
mol/L (Fig 1A and B). To investigate
whether Hx and TdR can protect cells from MTX-induced growth
inhibition, these metabolites were added to the medium. Hx (25 µmol/L) did not protect the cells from growth inhibition by high MTX
concentrations (>107 mol/L), but IC50
values increased fourfold and fivefold for HL60 and U937, respectively.
TdR (10 µmol/L) by itself increased the IC50 value of MTX
by 2.7-fold and 3.8-fold, respectively. In contrast to the addition of
Hx, TdR partly protected cells from high MTX concentrations (Fig 1A and
B). When Hx and TdR were added simultaneously, complete prevention of
the MTX effect was observed for U937 (Fig 1B). Also for HL60 cells, no
IC50 could be calculated (IC50 > 105 mol/L) (Fig 1A). To test the hypothesis that
compounds released by dying patient-derived ALL cells prevented
MTX-induced cell kill in the MTT-assay, lysed ALL cells were added to
HL60 and U937 cultures. IC50 values increased fourfold for
both cell lines (Fig 1A and B).
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| Fig 1.
Dose-response curves of MTX in absence or presence of 25 µmol/L Hx, 10 µmol/L TdR, or 1 × 106/mL lysed primary
ALL cells for (A) HL60 and (B) U937. The results are expressed as mean
values of three experiments ± SD. Protection from the MTX-induced
growth inhibition was most obvious when Hx and TdR were added
together.
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Lack of in vitro MTX cytotoxicity in patient-derived blast cells
using the MTT assay.
The effect of MTX was determined for blast cells of 83 leukemic
patients by the MTT assay. LC50 values could, however, not be determined in 90% of the samples because the leukemic cell survival
in the presence of MTX did not decrease below 50% of the control cell
survival. This lack of MTX cytotoxicity was observed even at very high
MTX concentrations up to 5.5 × 103 mol/L, as
shown for two patients in Fig 2. By applying the DiSC assay, also no dose-response curves were obtained (data not shown).
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| Fig 2.
Dose-response curves of MTX for samples derived from two
patients with ALL, in the absence and presence of TP (0.15 IU/mL) and
XO (0.02 IU/mL) (patient A) or in the absence and
presence of R75231 (1.65 µmol/L) (patient B). Both adaptations of the
MTT assay did not result in dose-response curves for MTX for 11 patient
samples.
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Prevention of rescue from MTX cytotoxicity.
The lack of MTX cytotoxicity on nondividing patient cells might be
associated with the presence of high concentrations of TdR and Hx.
Therefore, we determined the concentrations of these compounds in the
control wells of four patient samples after 4 days of
incubation. The concentration of Hx varied between 36.4 and 58.7 µmol/L, which was not increased relative to the
concentrations measured in the corresponding wells without cells
(Table 1). The TdR concentrations
ranged between <0.1 and 2.1 µmol/L, which is twice the
concentration measured in the absence of cells (Table 1). Preincubation
of the medium with TP and XO markedly reduced the TdR and Hx
concentrations in four samples measured (Table 1). However, this
reduction in the concentration of the metabolites did not result in
dose-response curves for 11 patient samples, as shown for a
representative sample in Fig 2. Cell membrane nucleoside transport was
inhibited by coincubation with R75231 to prevent protection of cells by
the presence of TdR in the medium. For 12 samples tested, R75231 did
not increase the cytotoxicity of MTX, as shown for one sample in Fig 2.
Optimization and validation of the in situ TSIA.
An alternative method to screen cells for antifolate resistance is
based on the inhibition of TS in intact cells.18,19,30 To
validate the TSIA, we determined antifolate sensitivity obtained with
the MTT assay and with the TSIA using the MTX-sensitive T-lymphoblastic leukemia cell line CCRF-CEM and three sublines, which are resistant to
MTX due to defective transport, impaired polyglutamylation, or elevated
DHFR. In addition to MTX, sensitivity to five other antifolates,
mentioned in the Materials and Methods section, was determined. These
compounds differ from MTX at the level of transport, polyglutamylation,
and/or target enzyme. A strong and significant correlation was
found between the IC50 values determined in the MTT assay
and both the TSI50,cont. (r = .89, P < .001, Fig 3) and the
TSI50,short (r = .66, P = .001) in the
TSIA. The Wilcoxon signed rank sum test showed that antifolate
sensitivity, as measured with the TSIA after continuous exposure, was
not different from antifolate sensitivity as measured with the MTT
assay (P = .69).
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| Fig 3.
Correlation between TSIA and antifolate growth inhibition
measured by the MTT assay for wild-type CCRF/CEM leukemia cells and
three MTX-resistant sublines. TSIA and MTT-based growth inhibition were
determined for MTX ( ) and five other antifolates (AG337, edatrexate,
GW1843U89, trimetrexate, and ZD1694) ( ). The
TSI50 values, measured by the 21-hour incubation TSIA, and
the IC50 values after 3 days of culture in the MTT assay
were significantly correlated (r = .89, P < .001).
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Because the original TSIA19 required 2 × 106 cells per drug per time point, we modified the assay by
testing different concentrations of drugs instead of different time
points. In addition, the substrate was changed from deoxyuridine to
deoxycytidine, which is more efficiently converted to dUMP, the direct
substrate for TS. The amount of 3H2O formed was
increased fourfold when deoxycytidine was used as a substrate instead
of deoxyuridine, reducing the amount of cells to 0.1 × 106 cells per drug per concentration. No difference in
TSI50 values was observed using these substrates as shown
in Fig 4A.
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| Fig 4.
(A) Comparisons of TSI50 (, TSI50,
cont.;  , TSI50, short) values obtained by the use
of [3H]-dUrd and
[3H]-dCyd as substrates in the in situ
TSIA. (B) Comparisons of TSI50 values (,
TSIcont.; , TSIshort) obtained in fresh
samples and after cryopreservation from 10 patients after 21 hours of
continuous MTX incubation and after a 3-hour incubation period followed
by an 18-hour drug-free period.
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Comparison of fresh and cryopreserved samples from 10 primary acute
leukemia patients showed that cryopreservation did not influence
MTX-induced TS inhibition during the continuous drug exposure
or the short drug exposure condition of the TSIA (Fig 4B). In
paired blast samples isolated from peripheral blood and from bone
marrow derived from seven patients, no differences in TSI50
values were observed (data not shown).
TSIA in T- and c/preB-ALL.
A large interpatient variation in MTX sensitivity was observed both in
TSI50,cont., ranging from 0.0067 to 0.76 µmol/L MTX and
in TSI50,short, ranging from <0.156 to >40
µmol/L MTX (Fig 5). The median
TSI50,cont. was not significantly different between 29 T-
and 51 c/preB-ALL samples (0.061 v 0.104 µmol/L MTX;
P = .46). The median TSI50, short, however, was
3.4-fold higher in T-ALL compared with c/preB-ALL (1.66 v 0.49 µmol/L; P = .001), but with a large overlap between both
groups (Fig 5).
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| Fig 5.
Concentrations of MTX necessary to inhibit 50% of the in
situ TS activity in T- and c/preB-ALL, measured after 21 hours
continuous drug exposure (TSI50,cont., left panel) or after
3 hours of drug incubation followed by an 18-hour drug-free period
(TSI50,short, right panel). Median values are represented
as a horizontal line.
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The role of MTX polyglutamates.
F-MTX is a nonglutamylatable analogue of MTX, with otherwise similar
characteristics.22 Therefore, by measuring the sensitivity to both MTX and F-MTX, more insight into the role of MTX polyglutamates would be provided. For F-MTX, no difference was observed for continuous exposure between T- and c/preB-ALL (median TSI50 values,
0.65 and 0.64 µmol/L, respectively; P = .68). The ratio
TSI50,cont. F-MTX/TSI50,cont. MTX was median 7 (range, 3 to 21), and the sensitivity for F-MTX was significantly
related to the sensitivity for MTX (r = .93; P < .001; Fig 6). After incubation in drug-free
medium, TS activity was fully recovered in the majority of the samples incubated with F-MTX, in contrast to the results obtained with MTX.
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| Fig 6.
Correlation between TSI50,cont values for MTX
and F-MTX determined with the in situ TS inhibition assay, in which
cells were incubated with MTX or with F-MTX for 21 hours. ( ),
c/preB-ALL; ( ), T-ALL.
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Because the difference in retention of TS inhibition between F-MTX and
MTX clearly indicated an important role for polyglutamylation of MTX,
we determined MTX accumulation and polyglutamylation for 47 samples.
TSI50,cont. and TSI50,short were both
significantly correlated with MTX accumulation
(Table 2). The ratio
TSI50,short/TSI50,cont., excluding the two
T- and the 16 B-lineage samples with
TSI50,short<0.156 µmol/L, was not correlated with
overall MTX accumulation, but rather with the percentage of MTX, which
had been converted to the pharmacologically more important
MTX-Glu4-6 (Table 2).
| |
DISCUSSION |
In this report, we showed that MTX resistance can be studied in primary
ALL cells by an in situ TSIA. Conventional total cell kill assays such
as the MTT assay9-12 and the DiSC assay13
cannot be used for this purpose due to salvage by purines and
thymidine, which protect cells from the cytotoxic effects of
MTX.26,31-34 A test to determine MTX resistance is
particularly important because MTX is an essential drug in the
treatment of ALL, and evaluation of MTX resistance in primary ALL
samples may improve the prognostic value of in vitro drug resistance
testing. In addition, research on clinically relevant mechanisms of MTX
resistance will be facilitated by the identification of resistant
subgroups of patient samples.
In the present report, we showed that Hx, TdR, and lysed ALL cells can
protect cell line cells against MTX-induced growth inhibition.
Protection by lysed ALL cells can (at least partly) be explained by the
release of metabolites such as Hx and TdR.16 In our
experiments, the concentrations of metabolites released by 1 × 106 lysed ALL cells/mL were high enough to increase
TSI50 values by fourfold. Higher concentrations of Hx (25 µmol/L) and TdR (10 µmol/L), however, were required for complete
protection of U937 cells against MTX-induced growth inhibition. Also
for HL60 cells, the simultaneous addition of Hx and TdR was more
efficient in protecting cells compared with the ALL lysate. In the
nonproliferating system of ALL lymphoblasts, however, the
concentrations of Hx and TdR released by spontaneously dying patient
cells may be sufficient to protect cells from MTX-induced death.
Because about 35% of the untreated ALL cells die spontaneously during
the 4 days of culture in the MTT assay,10 the subsequent release of Hx and TdR into the medium partly explains the observed lack
of MTX cytotoxicity in primary ALL cells obtained from patients. After
4 days of culture, we detected an increase in TdR concentration only
and not in Hx concentration in wells containing cells versus wells
without cells. This, however, might be a reflection of the consumption
of Hx by the remaining cells to survive the 4 days of culture. The
hypothesis that dying cells protect remaining cells from MTX
cytotoxicity is supported by several observations. The dilution of
plated ALL cells to less than 200 cells/well did result in a
dose-dependent effect of MTX, whereas in experiments with an initial
concentration of 10,000 cells/well, cells were protected from MTX
cytotoxicity.16 In another study with a MTX nonresponsive
xenograft, it was shown that incubation with XO and TP to degrade Hx
and TdR, respectively, did result in MTX-induced growth
inhibition.17
We adapted the MTT assay conditions to block potential rescue effects
by purine/pyrimidine salvage pathways. XO and TP indeed decreased the
concentrations of Hx and TdR in the medium, but still no dose-response
curves were observed for MTX on leukemic cell samples from 11 ALL
patients in the MTT assay. This result could imply that residual levels
of TdR and Hx were still sufficient to prevent the remaining patient
cells from MTX cytotoxicity. Moreover, other purines have been
described to rescue cells from MTX cytotoxicity such as guanosine and
adenosine.33 These metabolites are also degraded by XO, but
only after conversion to Hx. On the present chromatograms, these
metabolites could not be evaluated. Another explanation may be found in
an increase in extracellular folate pools due to the spontaneously
dying cells. However, these folates would prevent MTX toxicity by
competition for transport and polyglutamylation suggesting that higher
concentrations of MTX should overcome this rescue.
Coincubation of R75231, described to inhibit nucleoside transport of
Ehrlich ascites tumor cells,35 did not increase MTX cytotoxicity against ALL cells from patients. However, R75231 will only
inhibit the transport of nucleosides, while bases such as Hx are still
capable of entering the cell. In view of our cell line experiments and
data provided by others,26 it is clear that Hx alone cannot
fully rescue the remaining cells from MTX cytotoxicity. As already
described, replacing FCS by dialyzed FCS to remove folates,
nucleosides, and bases from the medium did not improve MTX
cytotoxicity, but decreased leukemic cell survival in
general.23 Altogether, it seems that different mechanisms of protection could be involved in patient-derived samples compared with proliferating cell lines.
Because no adaptations of the MTT assay resulted in dose-dependent MTX
cytotoxicity curves for primary ALL cells, we investigated an
alternative assay, described by others to detect antifolate sensitivity.18,36,37 In four cell lines and testing six
antifolates, a highly significant correlation between the 18-hour TSIA
and 3-day MTT assay suggested that the TSIA is a suitable antifolate sensitivity test for cell lines. For patient samples, replacing the
substrate deoxyuridine by deoxycytidine reduced the number of cells
needed, as tritium is released faster from deoxycytidine than from
deoxyuridine.19 This could be associated with the fact that
conversion of deoxyuridine to dUMP is catalyzed by the cell
cycle-dependent thymidine kinase I, which consequently has a low
activity in resting cells.38 Deoxycytidine is activated by
deoxycytidine kinase, which has a higher activity in leukemic cells and
is not cell cycle-dependent.38 The monophosphate
dCMP is efficiently deaminated to dUMP, the active
substrate for TS. This modification, together with an increase in
incubation times, resulted in an in situ MTX sensitivity assay, which
can routinely be performed on cryopreserved and on fresh ALL patient
cells and requires similar low numbers of cells as the conventional
total cell-kill assays.
Using the TSIA, samples from 86 children with ALL, both c/preB-and
T-ALL, were screened for MTX resistance. Both subtypes are treated with
MTX, but the outcome of T-ALL is worse in conventional therapy regimens
compared with c/preB-ALL as reviewed by Uckun et al.20 This
may be partly explained by resistance to MTX, as T-ALL displays a less
efficient MTX polyglutamylation compared with
c/preB-ALL.39,40 Although the TSIA might not always equate MTX-induced cytotoxicity, the relative in vitro MTX resistance for T-
versus c/preB-ALL is confirmed by the TSIA measurements. When cells
were allowed to efflux MTX during an 18-hour drug-free period after 3 hours of incubation, T-ALL samples were 3.4-fold more resistant to MTX
compared with c/preB-ALL cells (P = .001). This is in
accordance with the reported low accumulation of long chain
polyglutamates in T-ALL, which are preferentially retained inside the
cell.41
With the continuous 21-hour exposure condition, no difference in MTX
sensitivity between T- and c/preB-ALL could be detected. This suggests
that differences in polyglutamylation can be overcome during continuous
MTX exposure, which has also been described for cell
lines.29,42,43 This is supported by the experiments with
F-MTX, as the TSI50,cont. values for MTX were strongly
correlated with the TSI50,cont. values obtained for the
nonglutamylatable F-MTX.
Patient-derived leukemia samples, when continuously exposed to MTX for
21 hours, displayed TSI50 values varying almost 100-fold. High TSI50,cont. values might reflect MTX resistance, as
reviewed by Bertino44 due to (1) a defective transport
leading to a lower intracellular concentration of MTX, (2) a mutation
in DHFR, the main target enzyme, resulting in a low-affinity for MTX,
or (3) a higher level of DHFR. The relative chemoresistance in T-ALL may also be related to the more frequent prevalence of subclones with
elevated levels of DHFR in T-ALL compared with B-lineage ALL
samples.45 In addition, a human T-cell leukemia cell line was reported to contain twofold higher DHFR protein and mRNA levels compared with a human B-lineage leukemia cell line.46 The
TSIA, however, could not detect differences in MTX sensitivity between T- and c/preB-ALL samples in the continuous 21-hour exposure condition. This might be related to elevations in DHFR in subclones occurring in a
range of 10% to 90% of the cells; an overall activity assay would not
detect these differences. Moreover, the 21 hours of incubation in the
TSIA may overcome small differences in DHFR content. In addition,
several other factors involved in MTX cytotoxicity might obscure the
influence of only one parameter when measured by the end point TSIA.
The overall accumulation of MTX, as measured after 24 hours, is one
parameter of MTX resistance that is correlated with the MTX sensitivity
as measured with the TSIA. The comparison of a short (3 hours)
incubation followed by a drug-free period (18 hours) and a continuous
(21 hours) exposure condition may help to investigate intracellular
retention of MTX, possibly providing information on MTX
polyglutamylation defects. This hypothesis is supported by the
significant correlation found between the ratio of
TSI50,short/TSI50,cont. and the percentage MTX
present as MTX-Glu4-6.
In conclusion, the total-cell-kill MTT assay could not be adapted to
evaluate MTX cytotoxicity on patient-derived ALL samples. However, the
indirect TSIA based on MTX-induced TS inhibition proved to be
informative with respect to the extent of MTX sensitivity and
resistance. Using this assay, T-ALL samples were more MTX-resistant compared with B-lineage ALL samples after a 3-hour MTX exposure, followed by an 18-hour drug-free period; on long-term MTX exposure, both phenotypes were equally sensitive.
| |
FOOTNOTES |
Submitted July 29, 1998; accepted September 30, 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