1. No category

From www.bloodjournal.org by guest on November 7, 2014. For personal use only.
1994 84: 564-569
Differences in constitutive and post-methotrexate folylpolyglutamate
synthetase activity in B-lineage and T-lineage leukemia
JC Barredo, TW Synold, J Laver, MV Relling, CH Pui, DG Priest and WE Evans
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Differences in Constitutive and Post-Methotrexate Folylpolyglutamate
Synthetase Activity in B-Lineage and T-Lineage Leukemia
BY Julio
c. Barredo, Timothy W . Synold, Joseph
Laver, Mary V. Relling, Ching-Hon Pui, David
and William E. Evans
G. Priest,
Folylpolyglutamate synthetase (FPGS) is responsible for the
metabolism of natural folates and a broad range of folate
antagonists to polyglutamate derivatives. Recent studies indicated increased accumulation of methotrexate (MTX) polyglutamates (MTX-PG) in blast cells as a predictor of favorable treatment outcome in childhood acute lymphoblastic
leukemia (ALL). We determined the expression of FPGS activity in blasts from children with ALL at diagnosis and after
treatment with MTXas a single agent, before conventional
remission induction therapy. The levels of enzyme activity
in ALL blasts at diagnosis (median of 689 pmol/h/mg protein) were significantly higher ( P = .003) than those found
in acute nonlymphoblastic leukemia (ANLL) blasts (median
of 181 pmol/h/mg protein). Comparable lineage differences
in normal lymphoid versus nonlymphoid cells suggest a lineage-specific control of FPGS expression. FPGS activity increased in ALL blasts after in vivo exposure to MTX. The
median increase in FPGS activity was significantly higher ( P
= ,003)in B-lineage ALL (188%)than in T-lineage ALL (37%).
Likewise, the percentage of intracellular long chain MTX-PG
(G1u3-.J was significantly higher ( P = .02) in B-lineage ALL
(92%) than in T-lineage ALL (65%). consistent with higher
FPGS activity in B-lineage blasts. This finding could explain,
at least in part, the superior outcome in children with Blineage ALL treated with antimetabolite therapy.
0 1994 by The American Society of Hematology.
F
DHFR by MTX-PG. For the new generation of antifolates
aimed at TS (tomudex) and glycinamide ribonucleotide formyltransferase (lometrexol), polyglutamation not only results in prolonged intracellular retention
but also increases
their inhibitory activity against their target enzymes by more
than 100-fold.’~5 This effect is sufficiently pronounced that
these compounds have been considered prodrugs by some
investigators,‘.’ with FPGS being essential for their conversion to the active moiety. Furthermore, recent reports indicate low or altered FPGS activity as a mechanism of‘ resistance to cytotoxicantifolatesX and suggest a role in the
selectivity to antifolates by showing different kinetic behaviors of FPGS extracted from normal murine intestinal epithelium and from two antifolate sensitive mouse tumors.’
In thepresent study, we havedeterminedthelevels
of
FPGS activity in blasts from children with newly diagnosed
ALL treated with high-dose or low-dose MTX administered
asa single agent before conventionalremissioninduction
therapy. We found higher FPGS activity in ALL blasts when
compared with acute nonlymphoblasticleukemia (ANLL)
blasts, and this lineage difference in FPGS activity was also
found in normal lymphoid versus nonlymphoid hematopoieticprogenitors.Furthermore,
ourdata indicate that ALL
blast FPGS activity increases after in vivo exposure to MTX,
and that this increase is greater in B-lineage than in T-lineage
ALL.
OLATEANTIMETABOLITESareamongthe
most
widely used cancer chemotherapeutic agents. Methoof dihydrofolatereductase
trexate (MTX), aninhibitor
(DHFR), has played a significant role in the overall survival
and improved central nervous system (CNS) chemoprophylaxis of children with acute lymphoblastic leukemia
(ALL).’
More recently,a newgeneration of potentinhibitors of
ZD1694 [tomudex] and
thymidylate
synthase
(TS;
ie,
LY23 1514) and of de novo purinesynthesis via inhibition of
glycinamide ribonuclotide formyltransferase (ie, DDATHF
[lometrexol]) has been developed. These new classes of cytotoxic antifolates, and a wide variety of “classical” antifolates (ie, those with
a fused heterocyclic ring linked to p aminobenzoylglutamic acid), are substrates for the enzyme
folylpolyglutamate synthetase (FPGS).’ Several lines of evia central role in
dence indicate that polyglutamation plays
the therapeutic action of cytotoxic antifolates. After entering
mammalian cells, MTX, tomudex, and lometrexol are extensively converted to polyglutamate (PG) forms and as
such
are better retained inside the cell. For MTX, this prolonged
intracellular entrapment results in the extended inhibition of
From the Departments of Pediatrics and Biochemistry and Molecular Biology, Medical University of South Curolina, Charleston,
SC; the Pharmaceutical and Hematology-Oncology Departments, St
Jude Children’s Research Hospital, Memphis, TN; and the Departments ofPediatrics and Clinical Pharmacy, University of Tennessee,
Memphis, TN.
Submitted January 25, 1994; uccepted March 17, 1994.
Supported in purt by a University Research Council Grant from
the Medical University of South Curvlina and Leukemia Program
Project No. POI CA 20180, Cancer Center Core Grunts No. 21765
and R37-CA 36401, a Center of Excellence Grant from the State of
Tennessee. and the American Lebanese Syrian Associated Charities.
Address reprint requests to Julio C. Barredo, MD, Medical University of South Carolina, Pediatric Hematology-Oncology, I71 Ashley Ave, Charleston, SC 29425.
The publication costs of this article were defrayed in part by page
charge payment. This urticle must therefore be hereby marked
“advertisement” in uccordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/y4/8402-0029$3.00/0
564
MATERIALS AND METHODS
Materials. Sephadex G-SO (30 to X 0 pm), formaldehyde, ATP.
FdUMP,sucrose,HEPES,magnesiumchloride,EGTA,
benzamidine, soybean trypsin inhibitor, type II ovomucoid trypsin inhibitor,
monothioglycerol, and L-glutamic acid were purchased from Sigma
Chemical CO Inc (St Louis, MO). a-Toluenesulfonyl fluoride was
fromEastman Kodak CO (Rochester,NY).One-mililitersyringes
werefromBecton Dickinson (Rutherford,NJ).[3,4-’H]Glutamic
acid was purchased from New England Nuclear (Wilmington, DE)
and was used without further purification. The scintillation cocktail
used was Ready Protein+ from Beckman Instruments Inc (Fullerton.
CA). All mediaandserumwereobtainedfromGIBCOBRL
(Bethesda,
MD).
AntLCD34
(HPCA-I) and antiLCD10 (antifrom Becton Dickinson(San
CALLA)antibodieswerepurchased
Jose,CA).lmmunomagneticbeadscoated
with sheepantimouse
IgG1‘F‘’ (Dynabeads “450) were from Dynal Inc (Great Neck, NY).
Blood, VOI 84, NO 2 (July 15), 1994: Pp 564-569
From www.bloodjournal.org by guest on November 7, 2014. For personal use only.
POST-METHOTREXATEFPGSCHANGES
565
IN ALL
(6s)-Tetrahydrofolate was prepared from folic acid by an enzymatic
procedure described previously.lo Thymidylate synthase was prepared froma strain of Escherichia coli bearing a plasmid" that
allows expression of the Lactobacillus casei thymidylate synthase.
St Jude Children's Research Hospital (SJCRH) Total XIII therapeutic window. Children with ALL enrolled in SJCRH Total XIII
Protocol were treated with MTXonly during a 4-day therapeutic
window before the start of multiagent remission induction chemotherapy. This protocol was approved by the Institutional Review
Board and informed consent was obtained on study entry. Patients
were stratified according to age, presenting white blood cell count,
and DNA index, and then randomized to receive low-dose (LDMTX)
or high-dose MTX (HDMTX).I2 Patients randomized toLDMTX
received 180 mg/m2 MTX (30 mg/m2 orally [POI every 6 hours for
6 doses), whereas those on the HDMTX regimen were treated with
1,OOO mg/m? MTX intravenously (IV) over 24 hours (200 mg/m2
IV push, followed by 800 mg/m2 administered IV continuously for
24 hours). At 48 hours after initiation of MTX therapy, all patients
received leucovorin (LV) rescue (5 mg/m' IV or PO every 6 hours
for S doses) before starting remission induction chemotherapy. Bone
marrow samples for FPGS activity determinations were obtained at
diagnosis (0 hours) and before LV rescue ( 4 4 hours). For comparison, blasts from children withANLL obtained at diagnosis were
also assayed for FPGS activity.
Leukemic blasts cyrosol preparafion. Blasts were isolated from
bone marrow aspirates by a standardized technique using a FicollHypaque centrifugation gradient at 4°C. The mononuclear layer was
collected and washed three times in a culture media consisting of
90% RPM1 and LO% fetal bovine serum at 4°C. Then, two volumes
of homogenizing buffer, containing protease inhibitors as previously
described, were added." Cell suspensions were disrupted using an
Ultrasonics W-380 Sonicator (Heat Systems Ultrasonics, Farmingdale, NY). A 160,OOOg supematant fraction was then prepared using
a Beckman Airfuge driven at 30 psi for 18 minutes. This highspeed supernatant fraction was passed through a Sephadex G-50 spin
column (Sigma Chemical CO,St Louis, MO) to remove endogenous
glutamic acid that might interfere with the assay.
Hematopoietic cell isolation and cytosol preparation. After we
received approval from the Institutional Review Board and informed
consent was obtained, heparinized bone marrow was collected from
eight normal human volunteer donors. Mononuclear cells were separated using a Ficoll-Hypaque centrifugation gradient at 4°C. Cells
were then washed and incubated with mouse antihuman CD34 or
CD 10 monoclonal antibodies. Selected populations were isolated
using immunomagnetic beads coated with sheep antimouse IgGl'"'.
Finally, isolated cell populations were released from the immunomagnetic beads by the addition of chymopapain. After washing cells
with phosphate-buffered saline (PBS), high-speed supernatants were
prepared as described for leukemic blasts. This isolation was performed following a modification of the procedure described for immunomagnetic purging of neuroblastoma cells from humanbone
marrow.'4 The purity of all isolated cell populations was determined
byflow cytometric analysis. In addition, CD34' cells were plated
and cultured for determination of colony-forming unit granulocytemacrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and
mixed colonies (CFU-GEMM) using standard clonogenic assays.
FPGS microassay. This assay relies on the formation of a covalently bound ternary complex of TS, 5.10-methylenetetrahydrofolate
(or the polyglutamate forms of this compound), and fluorodeoxyuridylate (FdUMP), as previously described indetail.15Briefly, this
procedure involves two consecutive reactions. In the first reaction,
FPGS present in a sample catalyzes the addition of L-[3H] glutamic
acid to tetrahydrofolate forming ['H] tetrahydropteroyldiglutamate.
In a second reaction, the ['H] tetrahydropteroyldiglutamate is incorporated into a covalently bound ternary complex in the presence of
Table 1. FPGS Activity in Normal Hematopoietic Progenitors
Compared With Leukemic Blasts
____
~~
FPGS Activity* (prnollhlrng protein)
Cell Type
Normal hematopoietic progenitors
CDlO+/CD19'
;;;;3;;) ]
P
< .001
CD34'
Leukemic blasts at diagnosis
B-lineage ALL ( N = 24)
T-lineage ALL ( N = 13)
ANLL ( N
=
10)
801(203-3.584)]
261 (58-2.807)
181 (97-379)
p = ,003+
Abbreviation: N. number of patients from whom leukemic blasts
were isolated.
* Measurements of FPGS and isolationof cells as described in Materialsand Methods. For normal hematopoietic progenitors, median
and range were determined from three independent experiments.
t Denotes difference in FPGS activity in ALL (B- and T-lineage)versus ANLL blasts.
TS, formaldehyde, and FdUMP. The reaction mixture is then passed
through a Sephadex G-50 spin column to isolate macromolecular
complex from excess unreacted L-[3H] glutamic acid. The eluate is
collected into scintillation vials, and the radioactivity is determined
using a liquid scintillation counter. Protein concentrations were determined by the Bradford dye-binding procedure.''
MTX and M7X-PG assays. Bone marrow samples were obtained
44 hours after in vivo exposure to MTX and immediately placed on
ice. Leukemic blasts were isolated using a Ficoll-Hypaque gradient
at 4"C, and assays were conducted on freshly isolated blasts to
minimize drug efflux. MTX and invivo formation of MTX-PG were
determined using a combined high performance liquid chromatography (HPLC)-radioenzymatic assay as previously described."." This
two-step methodology consists of an HPLC separation, followed by
a radioenzymatic assay using DHFR asthe binding protein toquantitate MTX and MTX-PG in each HPLC fraction.
Statistical analysis. Interindividual comparisons of FPGS activity between various patient populations and sample times were made
using a Mann-Whitney U test, whereas intraindividual comparisons
were made using a Wilcoxon-Signed rank test. The percentage of
intracellular long chain MTX-PG in B- versus T-lineage ALL blasts
was compared using a Mann-Whitney U test.
RESULTS
Pafients studied. FPGS activity was measured in leukemic blasts obtained form 47 patients with ALL (34 B-lineage
and 13 T-lineage) and 10 with ANLL. Blasts were obtained
from all ANLL patients at diagnosis, before starting therapy.
In the 47 ALL patients, 20 had sufficient blasts for measurement of FPGS activity both at diagnosis and at 44 hours after
MTX therapy, whereas 17 werestudied only at diagnosis and
10 patients were studied only at 44 hours after starting MTX
therapy. There were sufficient blasts from the 44 hours bone
marrow for measurement of MTX-PG in 42 ALL patients
(31 B-lineage and 11 T-lineage).
FPGS activity in ALL and ANLL blasts. FPGS activity
in ALL (n = 37) blasts at diagnosis (both B-lineage and Tlineage) was significantly higher than in ANLL blasts (n =
10; median, 689 v 181 pmol/h/mg protein, P = .003; Table
1). Higher FPGS activity was seen in B-lineage blasts (CD10
or common ALL antigen [CALLA] positive) as compared
with T-lineage blasts (median, 801 v 261 pmol/h/mg protein;
From www.bloodjournal.org by guest on November 7, 2014. For personal use only.
566
h
c
.-
BARRED0 ET AL
p = 0.04
6000
0)
c
g 5000
a
-F
k
4000
:
-
3000
5
2000
after
activity
FPGS
blast
in
Changes
x
.c
2
1000
DX
LB-lineage
44h
DX
44h
L T-lineage
Table l), although this did not reach statistical significance
(P = .15) because of the heterogeneity of activity and small
sample size.
FPGS activity in normal hematopoieticprogenitors. NormalCDlO' cells, representative of lymphoid progenitors,
were isolated from human volunteer donors usingmonoclonal antibodies (MoAbs) and immunomagnetic beads (see
Materials and Methods). An 80% enrichment was confirmed
using MoAb staining and flow cytometric analysis, and this
population coexpressed the CD19 antigen (a B-lineage
marker throughout all stages of differentiation). For comparison with early nonlymphoid progenitors, an anti-CD34
MoAb was used following the same methodology. This partially purified population also coexpressed the CD33 antigen
(a myeloid marker) in 45% of isolated cells. As previously
reported, in normal human bone marrow, only 4.9% 0.8%
of mononuclear cells within the lymphoid-blast window
are CD34+.I9Additionally, less than 0.6% of B-lineage progenitors (CDlO/CD19+ cells) will coexpress the CD34 antigen, thus indicating negligible cross-reactivity.2o Normal
lymphoid progenitor cells (CDlO/CDl9+) expressed higher
FPGS activity when compared withnormal nonlymphoid
progenitor cells (CD34+; median, 828 v 158 pmoWmg protein, P < .001; Table 1). These results are consistent with
our finding of differences in expression of FPGS in leukemic
cell populations of lymphoid versus nonlymphoid origin.
Pre- andpost-MTX FPGS activity. Enzyme activity was
determined in leukemic lymphoblasts at diagnosis (0 hours)
and after exposure to single-agent MTX (44hours), but before leucovorin rescue. The subgroup of patients in whom
FPGSwas measured for the present study did not differ
significantly in presentation when compared with the overall
study patient population (data not shown). A median 2.4fold increase in FPGS activity was observed after exposure
to MTX when compared with pretreatment levels (P < .02;
Fig 1). FPGS ,activity increased in all patients studied, with
no apparent difference between patients treated with
HDMTX versus LDMTX.
FPGS activity in B-lineage versus T-lineage ALL. Although blast FPGS activity increased in all patients studied
at 44 hours post-MTX (Fig l), there was a significantly
Fig 1.
treatment with MTX. Activity in B-lineage
and (n = 14)
Tlineage (n = 6 ) lymphoblasts was measured at diagnosis immediately bdore MTX (DX) and 44 hours
after the start of MTX single-agent therapy ( 4 4 h).
Open symbols represent LDMTX and closed symbols
represent HDMTX patients;linesconnectpaired
samples for each patient; horizontal lines and numbars are median values. A median 2.4-fold increase
in post"TX 1 4 4 hours) ALL blast (BT-lineage)
and
FPGS activity was observed comparedwith diagnosis (DX, P < ,021.
greater fractional increase in blasts from patients withBlineage versus T-lineage ALL (median, 188% for B-lineage
v 37% for T-lineage blasts, P = ,003; Fig 2). Although there
was nota significant difference in activity between B-lineage
and T-lineage blasts obtained at diagnosis, there was significantly higher FPGS activity in B-lineage versus T-lineage
ALL blasts obtained at 44 hours post-MTX exposure (median, 1,249 v 308 pmoWmg protein, P = .03).
MTX-PG accumulation in B-lineage versus T-lineage
ALL. The percentage of intracellular long chain MTX-PG
(Glu3-6)was significantly higher ( P = .02) in B-lineage ALL
(92%) compared with T-lineage ALL (65%; Fig 3). This
finding is consistent with higher FPGS activity in B-lineage
versus T-lineage ALL blasts, andmayreflectthe
greater
fractional increase in post-MTX FPGS activity in B-lineage
compared with T-lineage ALL.
DISCUSSION
Tumor sensitivity and resistance to "classical" folate antimetabolites such as MTX and to the new generation of TS
p
x
-
0.003
36.5%
l
0'
I
T-lineage
B-lineage
Fig 2. Percentage change in FPGS activity from pretreatment (diagnosis) to 44 hours after MTX therapy in B-lineage (n = 141 versus
T-lineage (n = 6) blasts from children with ALL. Data were derived
from paired samples. Horizontal lines (and numberr) represent median percentage of change; boxes span 25th to 75th percentile for
patients with B-lineage (n = 14) and T-lineage (n = 6 ) ALL.
From www.bloodjournal.org by guest on November 7, 2014. For personal use only.
POST-METHOTREXATEFPGSCHANGES
100 r
80
(0
m
-
0
2
5
40-
* c
0
567
IN ALL
these differences between normal and transformed cells, we
isolated partially purified populations of normal bone marrow progenitor cells representative of lymphoid (CD101
CD19’) and nonlymphoid (CD349 lineages. Normal
lymphoid progenitor cells (CDlO/CDl9+) have high FPGS
activity, similar to enzyme activity in childhood ALL blasts,
which are CD10 (CALLA or common ALL) antigen positive
in 90% of cases.28In contrast, normal nonlymphoid progenitors (CD34+) express lower FPGS activity; likewise, in the
same range as those in ANLL blasts. It is unclear whether
these differences in FPGS activity found in normal and leukemic cells of lymphoid versus nonlymphoid origin reflect
changes in gene expression, or result from posttranscriptional or posttranslational modification(s). Nonetheless,
these differences suggest a cell lineage-specific control for
FPGS within the human lymphohematopoietic system, probably independent of the events leading to leukemogenesis.
The proliferation, differentiation, and survival of hematopoietic progenitor cells is sustained by a variety of hematopoietic growth factors. Even though little is known about the
precise cascade of events initiated by the binding of these
glycoproteins to their cell surface receptors, it is tempting
to speculate that they may also play a role in the lineagespecific control of FPGS. These data support the hypothesis
that the control of expression of FPGS is regulated by at
least two mechanisms, one of which is linked to proliferation 13-29 and another that controls enzyme levels during differentiation and is tissue (or cell lineage) specific.I3
When comparing blast cell FF’GS activity at diagnosis (0
hours) and after in vivo exposure to MTX (44 hours) in
children with ALL, we found a 2.4-fold increase in activity
( P < .02). The mechanism by which antifolates such as
MTX increase FPGS activity remains tobe defined, but
could involve translational and/or transcriptional modification(s) resulting in accumulation of the FPGS protein, as
was recently observed for thymidylate synthase invitro.”
Nimec and Galivan” reported a 1.5- to 2-fold increase in
the rate of MTX polyglutamation in folate-depleted H35
hepatoma cells in vitro, which maybe due to increased FPGS
activity or less competition by intracellular folates resulting
in a consequent increase of FPGS sites available for MTX
binding. Alternatively, drug-induced or nutritional folate deficiency might upregulate the expression of FPGS as a compensatory mechanism to insure cellular integrity. In addition,
MTX-induced alterations in cell cycle regulation andor
quantitative changes in the proliferative cell fraction (percentage of S-phase cells) in the bone marrow could result
in the changes in FPGS activity detected at 44 hours (postMTX).
Children with B-lineage ALL have a better prognosis than
both children with T-lineage ALL and adults with B- or Tlineage ALL. Goker et a132reported higher in vitro accumulation of total MTX and MTX-PG in pediatric B-lineage blasts
than in blasts from T-lineage ALL patients and adults with
B-lineage ALL.They suggested decreased polyglutamate
formation or increased breakdown as responsible for the
reported low levels of MTX-PG in T-lineage and adult Blineage ALL. Our results indicate that blasts from children
with B-lineage ALL tend to have higher constitutive FPGS
t
T-lineage
B-lineage
Fig 3. Percentage of intracellular MTX metabolized to long chain
polyglutamates (MTX-Po,)
in vivo, in patients with B-lineage (n =
31) and T-lineage(n = 11) ALL. Horizontal lines land numbers) represent medianvalues; boxes span 25thto 75th percentile. Blasts were
obtained U hours after starting therapywith MTX and before leucovorin rescue ( w e Materials and Methods for details).
inhibitors (ie, tomudex, LY231415) and GARFT inhibitors
(ie, lometrexol) is highly dependent on polyglutamation by
FPGS.2-5To our knowledge, this is the first study to characterize the in vivo differential changes in blast FPGS activity
that occur after MTX therapy in children with B-lineage and
T-lineage ALL.
Our findings indicate that lymphoblasts from most children with ALL at diagnosis express high in vivo FPGS activity, providing a biochemical basis for their intrinsic sensitivity to antifolates, ie, MTX. Moreover, Whitehead et a12’
showed a survival advantage for children with ALL whose
blasts at diagnosis accumulated higher levels of MTX and
MTX-PG in vitro. Our study provides one mechanism by
which higher MTX-PG accumulation would occur in this
patient population, ie, higher ALL blast FPGS activity. In
contrast, MTX has proven to have no significant therapeutic
activity in ANLL.22Whitehead et alZ3 suggested increased
MTX-PG catabolism, whereas Lin et
reported normal
MTX transport and suggested decreased FPGS activity leading to decreased long chain MTX-PG accumulation in ANLL
blasts as a mechanism for resistance in vitro. Our data indicate that ANLL blasts have low FPGS activity, suggesting
decreased FPGS as a mechanism for their lower accumulation oflong chain MTX-PG and a biochemical basis for their
resistance to antifolates. Thus, the significant differences in
FPGS activity in ALL versus ANLL blasts could provide a
biochemical basis for their differential intrinsic sensitivity
to antifolates.
The expression of FPGS activity in normal and malignant
tissues appears to play a critical role in the sensitivity and
selectivity to a n t i f o I a t e ~ . * * Barredo
~ ~ . ~ ~ . ~and
~ MoranJ3previously reported undetectable levels of FPGS activity in circulating mature hematopoietic cells, whereas enzyme activity was found to be high in blasts from adult and pediatric
ALL patients. In a related study, Koizumi et a127reported
very low in vitro accumulation of MTX-PG in an unpurified
population of normal human bone marrow cells. To address
From www.bloodjournal.org by guest on November 7, 2014. For personal use only.
568
activity, but, more importantly, have a significantly higher
increase in their FPGS activity after in vivo exposure to
MTX when compared with blasts from children with Tlineage ALL ( P = .003). Furthermore, B-lineage ALL blasts
accumulated a higher percentage of long chain MTX-PG
compared with T-lineage ALL blasts (P = 0.02), consistent
with higher FPGS activity in B-lineage versus T-lineage
ALL. Long chain PG are preferentially retained inside the
cell; thus, drug efflux would be expected to have negligible
effects in the analysis of our data. However, it remains to
be determined whether multiple mechanisms (eg, transport,
gamma-glutamyl hydrolase, intracellular folate content, and
FPGS) contribute to the lineage differences in MTX-PG accumulation. These long chain MTX-PG are considered to
be the active metabolites and responsible for enhancing the
cytotoxicity of the parent compound.6,26Our invivo data
provide a potential basis for the favorable response of pediatric B-lineage ALL patients to MTX containing treatment
regimens. These results and future studies addressing lineage-specific regulation of FPGS may provide a biochemical
basis for determining specific leukemic phenotypes responsive to MTX and other agents that are substrates for FF'GS.
BARRED0 ET AL
in CCRF-CEM cells after short-term, high-dose treatment with this
drug. Cancer Res 48:2149, 1988
9. Rumberger BG, Barrueco JR, Sirotnak FM: Differing specificities for 4-aminofolate analogues of folylpolyglutamyl synthetase
from tumors and proliferative intestinal epithelium of the mouse
with significance for selective antitumor action. Cancer Res 50:4639,
I990
IO. Moran RC, Spears CP, Heidelberger C: Biochemical determinants of tumor sensitivity to 5-fluorouracil: Ultrasensitive methods
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76:1456,
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I I . Pinter K, Davisson VJ, Santi DV: Cloning, sequencing and
expression ofthe Lacrobncillus casei thymidylate synthase gene.
DNA 7:235, 1988
12. Synold T, Relling M, Boyett J, Rivera G, Sandlund T, Pui
C-H, Crist W, Evans W: In vivo intracellular concentrations of methotrexate polyglutamates (MTX-PG) in acute lymphocytic leukemia
(ALL) blasts from children randomized to receive either high-dose
(HDMTX) or low-dose (LDMTX) methotrexate. Proc Am SOCClin
Oncol 12:131, 1993 (abstr)
13. Barredo J, Moran RC: Determinants of antifolate cytotoxicity:
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14. Worthington-White DA, Gee AP, Gross S (eds): Advances
ACKNOWLEDGMENT
in Bone Marrow Purging and Processing. New York, NY. WileyLiss, 1992, p 147
The authors thank Elizabeth Willits, Yagin Chu, and Emily Melton
15. Antonsson B, Barredo J, Moran RC: A microassay for mamfor their excellent technical assistance, and the SJCRH Cell Bank
malian folylpolyglutamate synthetase. Anal Biochem 186:8, 1990
for its support of this project. We also thank Dr Richard G. Moran
16. Bradford MM: A rapid and sensitive method for the quantitafor helpful discussions during preparation of this manuscript.
tion of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal Biochem 72:248, 1976
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