Clinical Cancer Research Vol. 8, 2423-2429, July 2002
© 2002 American Association for Cancer Research
Experimental Therapeutics, Preclinical Pharmacology |
Methotrexate Intracellular Disposition in Acute Lymphoblastic Leukemia
A Mathematical Model of 
-Glutamyl Hydrolase Activity1
John Carl Panetta,
Amelia Wall,
Ching-Hon Pui,
Mary V. Relling and
William E. Evans2
Departments of Pharmaceutical Sciences [J. C. P., A. W., M. V. R., W. E. E.] and Hematology-Oncology [C-H. P.], St. Jude Children’s Research Hospital, Memphis, Tennessee 38105-2794, and Colleges of Pharmacy and Medicine, University of Tennessee, Memphis, Tennessee 38101 [C-H. P., M. V. R., W. E. E.]
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ABSTRACT
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Methotrexate (MTX) is an antifolate that is widely used for the treatment of childhood acute lymphoblastic leukemia (ALL) and a number of other malignant and nonmalignant diseases. Within cells, MTX is metabolized to more active methotrexate polyglutamates (MTXPG), and these polyglutamates are subsequently cleaved in lysosomes by 
-glutamyl hydrolase (GGH). GGH is reported to act as either an endopeptidase or an exopeptidase, exhibiting species differences in these functions. To better define the in vivo functions of GGH in human leukemia cells, we characterized GGH activity with different MTXPG substrates (MTX with three to five glutamates) in human T- and B-lineage leukemia cell lines, and in primary leukemia cells from newly diagnosed patients with ALL. Parameters estimated from fitting a series of hypothetical mathematical models to the data revealed that the experimental data were best fit by a model where GGH simultaneously cleaved multiple glutamyl residues, with highest activity at cleaving the outermost or two outermost residues from a polyglutamate chain. The model also revealed that GGH has a higher affinity for longer chain polyglutamates. Together, these findings provide new insights to the intracellular disposition of MTX in human ALL cells, and provides a mechanism-based model for characterizing differences among patients and genetic subtypes of ALL.
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INTRODUCTION
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Childhood ALL3
is one of the most common cancers in children. Despite ALL cure rates >75%, a substantial number of children continue to relapse after modern treatment regimens (1)
. MTX, an antifolate, is used in essentially all of the contemporary treatment protocols for childhood ALL. MTX and to a greater extent its active metabolites, MTXPG, inhibit several enzymes that are essential for cellular folate homeostasis, including dihydrofolate reductase, thymidylate synthase, glycinamide ribonucleotide transformylase, and aminoimidazole carboxamide ribonucleotide transformylase (2
, 3)
, thereby impairing purine, pyrimidine, and DNA synthesis. Additionally, long chain polyglutamates (MTXPG4–7) are more avid inhibitors of these target enzymes and are also retained within cells for a longer period of time, thereby prolonging their antifolate effects (4
, 5)
. Higher accumulation of MTXPG has been associated with increased cytotoxicity and greater antileukemic effects in childhood ALL (4
, 6)
.
Differences in intracellular MTXPG accumulation have been observed among specific subtypes of childhood ALL. In particular, T-lineage ALL has been shown to have lower accumulation of MTXPG compared with B-lineage ALL [median of 552 pmol/109 cells (2.2 µM)4
for T-lineage compared with 1413 pmol/109 cells (5.5 µM)4
for nonhyperdiploid B-lineage and 3371 pmol/109 cells (13.5 µM)4
for hyperdiploid B-lineage ALL, with a dose of 1 g/m2 given i.v. over 24 h; 7
, 8
], which is attributable in part to lower FPGS in T-lineage ALL (8
, 9)
. Understanding the intracellular dynamics of MTX and its active metabolites may provide important insights to the observed differences in MTXPG accumulation and pharmacological effects. MTXPG is formed by the sequential addition of up to seven glutamyl residues, catalyzed by cytosolic FPGS. These glutamyl residues are also subsequently cleaved from MTXPG by GGH, after transport of MTXPG into the cellular lysosome. In vitro studies have shown that increased levels of GGH activity are associated with lower MTXPG accumulation and decreased effects of MTX (10
, 11)
. Although, ectopic overexpression of very high levels of GGH did not perturb MTX sensitivity in a human fibrosarcoma (HT-1080) and a human breast carcinoma (MCF-7) cell line (12)
. However, clinical studies (9
, 13
, 14)
have demonstrated that MTXPG accumulation in primary leukemia cells from patients could be predicted by the ratio of FPGS:GGH activity.
Several studies have characterized the activity of human and rat GGH in vitro reporting that rat GGH acts solely through endopeptidase, whereas human GGH produced MTXPG1–4 from MTXPG5, primarily through exopeptidase activity (15)
. Furthermore, Rhee et al. (16)
and Galivan et al. (11)
demonstrated that human GGH only cleaves the outermost or two outermost glutamates, favoring cleavage of two glutamate residues. To develop a better mechanistic understanding of human GGH activity, we developed a set of mathematical models to test experimentally developed hypotheses on the activity patterns of GGH in human leukemia cell lines and in primary leukemia cells from patients with ALL, providing new insights to the function of this enzyme.
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MATERIALS AND METHODS
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Experimental Methods.
The human leukemia cell-lines CEM (T-lineage) and Nalm-6 (B-lineage) were maintained in RPMI 1640 with 10% fetal bovine serum and 1% glutamine at 37°C. Primary leukemia cells were also isolated from peripheral blood of patients with ALL (10 female/13 male, median age 7.2 years, 18 B-lineage, 5 T-lineage). Informed consent for an Institutional Review Board-approved research protocol was obtained from the parents of the patients. To measure GGH activity, cells were washed twice in buffer (12.8 g sucrose and 373.9 mg HEPES), then resuspended in buffer (1 x 107 cells/ml) and 10% Triton X-100 to a 0.1% final concentration. The protein content of the extract was determined using the Bio-Rad protein assay. The GGH reaction mixture contained 40 µg protein and 50 µM MTXPGi (i = 3... 5), in 200 µL of MTEN buffer [50 mM 4-morpholinepropanesulfonic acid; 2-(N-morpholino)ethanesulfonic acid, 25 mM Tris-base, 25 mM ethanolamine, 100 mM NaCl, 1 mM ZnCl2, and 2 mM DTT], with a final pH of 4.5 and was incubated for five different time intervals (1, 4, 22, 48, and 72 h) at 37°C in the two cell lines and 1 patient with sufficient cells for multiple incubation times, and for 1 h at 37°C in the remaining 22 patient samples. The reaction was stopped by heating the samples at 95°C for 5 min. Samples were cooled on ice for 15 min then centrifuged (11,000 x g; 5 min; 4°C), and stored at 4°C. MTXPG2–5, and MTX were isolated and quantified using reverse phase high-performance liquid chromatography with UV (309 nm) detection (7)
. All of the compounds were isolated from a C18 column with mobile phase A [methanol, 5 mM sodium phosphate, and 2.5 mM tetra butyl ammonium nitrate (pH 7.3); 20:80, v/v] and mobile phase B [methanol, 5 mM sodium phosphate, and 2.5 mM tetrabutyl ammonium nitrate (pH 7.3); 40:60, v/v]. A linear gradient from 45% mobile phase B to 100% mobile phase B was used over 30 min, with a flow rate of 1.3 ml/min. All of the data were collected by Shimadzu Class-VP software. To demonstrate the stability of the MTXPG substrates, MTXPG2–5 were individually incubated for both 24 and 72 h at 37°C with all of the above reaction conditions, except proteins had been denatured by heating to 95°C for 5 min. The results were then analyzed by high-performance liquid chromatography as described above. The recovery of MTXPG2–5 was 88–100% (median of 93.5%) of the starting substrate amount for the 24 h incubation and 84–104% (median of 91%) of the starting substrate amount for the 72 h incubation.
GGH activity was determined after incubating the substrate (MTXPG3, MTXPG4, or MTXPG5) for 1 and 4 h in the two cell lines, and for 1 h in the patient samples by measuring the total amount of product formed per µg protein per h.
Model Development.
The GGH enzyme reaction is described in Equation 1
, where MTXPGi is the concentration of the polyglutamate with i glutamyl residues, GGH is the concentration of the enzyme, 
and is the enzyme bound complex:
The general mathematical form, describing the enzyme reaction, is:
where MTXPGi, i = 1···5, is the pmol amount of MTXPGi, MTXPG1 = MTX, and
are the GGH cleavage rate parameters from MTXPGi to MTXPGj, i > j, and are functions of MTXPGi, e.g., k54 is the rate of GGH cleavage from MTXPG5 to MTXPG4, and t is time in h. Vmaxi
j, the maximum hydrolysis rate from MTXPGi to MTXPGj (pmol/h), and Kmi, the 50% maximum hydrolysis rate of MTXPGi (pmol), are defined as follows:
where r is the concentration of enzyme and the 
’s are defined in Equation 1
.
Three potential variations of the above model were fit to the data to explore alternative mechanisms of GGH cleavage and MTXPG disposition:
(a) For Model 1, on the basis of results in (15)
, only the outer cleavage rate parameters are nonzero (exopeptidase), i.e., k53 = k52 = k51 = k42 = k41 = k31 = 0;
(b) For Model 2, on the basis of results in (16)
, only the outer two cleavage rate parameters are nonzero, i.e., k52 = k51 = k41 = 0; and
(c) For Model 3, all of the rate parameters (kij) are nonzero—cleavage at any bond.
Parameter Estimation.
Maximum likelihood estimation as implemented in the ADAPT II software (17)
was used to estimate model parameters using all five of the incubation times. Initial parameter estimates were determined from previously published studies of GGH activity (9
, 11
, 13
, 18)
. The SS and the AIC were used as measures of accuracy and parsimony of the different models, and precision of the parameter estimates was assessed by the CV. The t test was used to determine significance between parameter estimates. This was based on ADAPT II-generated SE estimates for the parameters.
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RESULTS
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Mean GGH activities, determined after the 1 and 4-h (when available) incubations with substrate, are provided in Table 1
. GGH from the T-lineage CEM cell line hydrolyzed MTXPG at a higher rate (as much as 2.75-fold higher; P < 0.01) compared with GGH derived from B-lineage NALM6 cells. GGH activity in primary leukemia cells obtained from newly diagnosed patients was comparable with activity in the cell lines (Table 1)
with a trend toward higher GGH in T-lineage ALL. Over a 72-h incubation, when MTXPG5 was used as the substrate, 91%, 51%, and 47% was hydrolyzed to MTXPG1 in CEM, NALM6, and the primary ALL blasts,5
respectively.
Comparison of Models.
The three structural models were fit to these data (i.e., the results from the 1, 4, 22, 48, and 72 h incubations), and the resulting SS and AIC for each model are summarized in Table 2
. Figs. 1
and 2
depict the fit of each of the three models to the primary data. The high SS and AIC with Model 1 relative to the other models indicate that GGH does not act solely as an exopeptidase (Fig. 2, A, C, and E)
. Model 2, which assumes that GGH simultaneously cleaves a maximum of two bonds, gave a better fit of the data than Model 1 (Fig. 2, B, D, and F)
, but the higher SS and AIC of Model 2 made it inferior to Model 3. Model 3 gave the best fit to the data (Fig. 1)
based on the 6–5800-fold smaller SS and AIC (see Table 2
). The estimated parameters for Model 3 are summarized in Table 3
, and the superior fit of the data by Model 3 indicates that GGH cleaves at all of the bonds and not just the outer one or two bonds.
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Fig. 1. Fit of best model (Model 3) to data with MTXPG5 used as the substrate. The data are indicated by:  , MTXPG5;  , MTXPG4;  , MTXPG3; +, MTXPG2; and •, MTXPG1. The model fit is indicated by: ——, MTXPG5; ———, MTXPG4; —, MTXPG3; — - —, MTXPG2; and · · · ·, MTXPG1. A, CEM cell line; B, NALM6 cell line; and C, primary ALL blasts.
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Fig. 2. Fit of alternative models (Models 1 and 2) of GGH cleavage with MTXPG5 used as the substrate. The data are indicated by: , MTXPG5; , MTXPG4; , MTXPG3; +, MTXPG2; and •, MTXPG1. The model fit is indicated by: ——, MTXPG5; ———, MTXPG4; —, MTXPG3; — - —, MTXPG2; and · · · ·, MTXPG1. A, Model 1 fit to CEM; B, Model 2 fit to CEM; C, Model 1 fit to NALM6; D, Model 2 fit to NALM6; E, Model 1 fit to primary ALL blasts; and F, Model 2 fit to primary ALL blasts.
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Differences in Cleavage Patterns.
Table 4
summarizes differences in the maximum rate parameters (Vmaxij) for GGH cleavage of MTXPG3, MTXPG4, or MTXPG5 in the CEM (human T-lineage), NALM6 (B-lineage), and primary leukemic blasts (early pre-B-lineage).4
In all three of the cell types, GGH favored cleavage of the outer-most bond of MTXPG4 and MTXPG5. In addition, cleaving either the outer-most or the two outer-most bonds was the most dominant cleavage pattern for both CEM and NALM6 cell lines (there were not sufficient cells to assess multiple substrates in the primary ALL cells), as indicated by the significant (P < 0.01) fold difference for Vmax54 to Vmax52, Vmax54 to Vmax51, and Vmax43 to Vmax41 (Table 4)
. Also, both CEM and NALM6 cells favored cleavage of the two outer-most bonds over only the outer-most bond of MTXPG3, as indicated by the significant (P < 0.01) fold differences for Vmax32 to Vmax31. Finally, the affinity for GGH decreased (i.e., an increase in Kmi) with shorter chain polyglutamates in both CEM and NALM6 cell lines, as seen by the large fold increases for Km4 to Km5 and Km3 to Km5 in Table 4
.
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Table 4 Differences in cleavage rate and affinity constants for GGH cleavage in human leukemia cells. The values indicate the fold difference. All of the differences are significant with P < 0.01
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Differences between Cell Lines.
To explore potential lineage differences in MTXPG hydrolysis by GGH, cleavage rates were compared between the cell lines. The maximum rate of GGH cleavage of MTXPG5 to all of the products (Vmax54,3,2,1 = 
i=14 Vmax5i) was 3.2-fold higher in the CEM cell line compared with NALM6 cell line (P < 0.01) and 2.8-fold higher in the primary ALL blasts (4)
when compared with the NALM6 cell line (P < 0.01). Also, the affinity was significantly different in the primary leukemic cells (i.e., Km5 was 5.9-fold lower in CEMs and 5.5-fold lower in NALM6; P < 0.01 compared with the Km5 in primary ALL blasts).4
These differences led to a significantly higher intrinsic GGH activity with MTXPG5 as the substrate (Vmax54,3,2,1/Km5) in the T-lineage CEM cell line compared with the B-lineage cell lines (3.5-fold when compared with NALM6 and 6.7-fold when compared with the primary B-lineage ALL blasts.)5
Similarly, the maximum rate of cleavage for MTXPG4 (Vmax43,2,1) was 1.9-fold higher (P < 0.01) in the CEM cells compared with the NALM6 cells, with comparable affinities. The T-lineage CEM cells had greater overall hydrolysis of MTXPG compared with the B-lineage NALM6 and the primary leukemic cells from a patient with early pre-B-lineage ALL.
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DISCUSSION
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Several studies have measured GGH activity in vitro. Longo et al. (13)
estimated GGH-specific activity to be between 3.2 and 47.4 (pmol/h/µg protein) in primary leukemia cells (ALL) using MTXPG5 as a substrate. These results are consistent with the value of 10.8 pmol/h/µg protein we obtained in primary leukemia cells in the current study. Furthermore, it was observed by Samuels et al. (18)
, using MTXPG4 as the substrate, that the estimates for Vmax43,2,1 (pmol/h/µg protein) and Km4 (µM) in mouse L1210 leukemia cells were 0.702 and 28.3 ± 3.4, respectively, and in small intestine the estimates were 7.74 and 10.1 ± 1.5, respectively. These results are similar to our experimentally estimated Vmax43,2,1 and Km4, when Km4 is converted to units of µM, (i.e., Km4 = 9.4 µM for the CEM cell line and Km4 = 5.9 µM for the NALM6 cell line).
By considering several variations of the potential mechanistic mathematical model of GGH activity, we were able to test several hypotheses about MTXPG cleavage by GGH. The superior fit of Model 3 indicates that GGH cleaves at all of the bonds not just the outer-most or two outer-most -glutyamyl bonds. This differs from Rhee et al. (16)
and Yao et al. (15)
, who report that, at most, only the outer two bonds are cleaved. Rhee et al. (16)
also reported that the cleaving of two glutamate residues at a time was more common than the cleaving of one. Our experiments also demonstrated the preference to cleave two glutamate residues over one but only for MTXPG3 in both the CEM and NALM6 cell lines. In contrast, our experiments showed that GGH preferred cleaving the outer bond on MTXPG5 in all of the cells considered, and also on MTXPG4 in the CEM and NALM6 cell lines (5)
. We also found that GGH activity has a higher affinity for longer chain polyglutamates in both CEM and NALM6 leukemia cells (5)
. Finally, we found significant differences in GGH activity among the two different leukemia cell lines and the primary leukemia cells. In particular, for long chain polyglutamates (MTXPG4 and MTXPG5), there was significantly greater hydrolysis in the T-lineage CEM cell line compared with the B-lineage cell lines. This is consistent with previous reports from our lab and others (6, 7, 8)
of lower accumulation of MTX polyglutamates in T-lineage ALL compared with B-lineage ALL. However, there was not a significant difference in the hydrolysis of short chain polyglutamates (MTXPG3) in the CEM and NALM6 cells (5)
. This suggests that when making comparisons between lineages, results may differ if a short chain or long chain polyglutamate is used as the substrate, and must be considered when interpreting studies that failed to show a difference in GGH activity between B- and T-lineage ALL (9)
. Interestingly, our prior studies revealed higher FPGS activity in NALM6 compared with CEM (19)
, similar to lineage differences in FPGS activity in primary ALL blasts (8)
. Higher FPGS activity in B-lineage NALM6 cells, coupled with lower GGH activity as observed in the current study, provide a dual mechanism for higher accumulation of MTXPG in this B-lineage ALL cell line. However, there appears to be considerable heterogeneity in GGH activity in ALL blasts from different patients (9)
,6
and additional studies are needed to determine whether there are significant lineage differences in GGH activity among patients with ALL. It is also likely that multiple mechanisms will be involved in determining the rate of MTXPG hydrolysis in ALL blasts (e.g., lysosomal transporters, GGH protein expression in lysosomes, and so forth), such that a number of mechanisms may regulate net GGH hydrolysis of folates and antifolates in leukemia cells. However, currently there are no polymorphisms in the human GGH gene that are known to alter GGH function. Likewise, we have sequenced the GGH gene locus from three patients and no single-nucleotide polymorphisms were found.5
Clearly, it will be important to investigate germ-line polymorphisms in the future to determine whether functionally important single-nucleotide polymorphisms exist in the human GGH gene. Going forward, it will be important to additionally define cellular and molecular determinants of GGH activity in leukemia cells and normal tissues, to elucidate strategies for overcoming this potential mechanism of MTX resistance.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by Cancer Center CORE grant CA21765, NIH R01 CA78224, by a Center of Excellence Grant from the State of Tennessee, and by the American Lebanese Syrian Associated Charities (ALSAC). 
2 To whom requests for reprints should be addressed, at St. Jude Children’s Research Hospital, 332 North Lauderdale St., Memphis, TN 38105-2794. Phone: (901) 495-3663; Fax: (901) 525-6869; E-mail: william.evans{at}stjude.org
3 The abbreviations used are: ALL, acute lymphoblastic leukemia; MTX, methotrexate; MTXPG, methotrexate polyglutamate; GGH, -glutamyl hydrolase; FPGS, folylpolyglutamate synthetase; SS, sum of squares; AIC, Akaike Information Criterion; CV, coefficient of variation.
4 Obtained using the conversion 250 fl/cell of intracellular volume.
5 From one patient with sufficient blasts.
6 W. E. Evans, unpublished observations.
Received 12/21/01;
revised 3/19/02;
accepted 3/20/02.
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ABSTRACT
INTRODUCTION
