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Vol. 62, Issue 1, 102-109, July 2002
Leukaemia Research Fund Molecular Pharmacology Specialist Programme, Cancer Research Unit (S.A.C., L.A.H., M.L., E.C.M., L.M., A.G.H.), and Medical Molecular Biology Group (C.P.F.R.), Medical School, Newcastle University, Newcastle-upon-Tyne, UK
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Although the thiopurine drugs 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are well established agents for the treatment of leukemia, controversies remain regarding their main mode of action. Previous evidence has suggested that although 6-TG exerts a cytotoxic effect through incorporation of 6-thioguanine nucleotides into newly synthesized DNA (DNA-TGN), an important component of the mode of action of 6-MP is inhibition of purine de novo synthesis (PDNS) through the production of S-methyl-thioinosine 5'-monophosphate (MeTIMP), not formed in cells exposed to 6-TG. We have shown that thiopurine methyltransferase (TPMT) modulates this effect. By transfection of the human TPMT gene using an inducible system to produce a 3.8-fold increase in TPMT activity in the ecdysone receptor 293 embryonic kidney cell line, we demonstrated a 4.4-fold increase in sensitivity to 6-MP. This was associated with a rise in intracellular levels of MeTIMP but a decrease in levels of DNA-TGN. In contrast, induction of TPMT produced a 1.6-fold decrease in sensitivity to 6-TG, a decrease in levels of DNA-TGN, and an increase in levels of methylated thioguanosine monophosphate. Exposure of cells to equitoxic doses of drug showed similar incorporation of DNA-TGN for 6-TG but for 6-MP significantly reduced DNA-TGN in TPMT-induced compared with uninduced cells. For equitoxic doses of 6-MP, equivalent levels of MeTIMP correlated with equivalent amounts of PDNS. These observations suggest that intracellular TGN levels do not give an accurate reflection of cytotoxic potential in patients treated with 6-MP, because different levels of DNA-TGN may be associated with equitoxic effects.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Since
their introduction into clinical practice more than 4 decades ago, the
purine analogs 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) have
been used extensively in the treatment of acute leukemia. 6-MP has been
preferred over 6-TG (mostly for reasons of custom and practice) during
continuing (maintenance) therapy in childhood acute lymphoblastic
leukemia (ALL), whereas 6-TG has been used during consolidation and in
remission induction in acute myeloid leukemia. However, several recent
trials have sought to establish whether 6-TG might be a more effective
agent than 6-MP during continuing therapy (Erb et al., 1998
; Lancaster
et al., 1998), including the present Medical Research Council ALL 97 trial. One rationale for these studies has been the observation that
treatment with 6-TG results in higher levels of thioguanine nucleotides (TGNs) within erythrocytes, which are used as a surrogate for levels in
leukemic cells. It has been assumed that the TGN level correlates with
higher levels of incorporation of fraudulent nucleotides into DNA and
therefore higher levels of cell kill (Maddocks et al., 1986).
The assumption that TGN levels are a direct measure of cytotoxicity
ignores the significant differences that exist between the
intracellular metabolism of the thiopurine drugs. Both 6-MP and 6-TG
are prodrugs that require activation by hypoxanthine-guanine phosphoribosyl transferase (HGPRT; Fig.
1) to exert a cytotoxic effect (Bertino,
1991). Competing with HGPRT for the metabolism of 6-MP are three
enzymes: thiopurine methyltransferase (TPMT), aldehyde oxidase, and
xanthine oxidase. In the case of 6-TG, xanthine oxidase can metabolize
the drug only after prior conversion by guanase. The products of these
competing reactions produce metabolites with little or no cytotoxic
activity. Metabolism of 6-TG by HGPRT produces 6-thioguanosine
5'-monophosphate (TGMP), which is further metabolized by a series of
kinases and reductases to produce deoxy-6-thioguanosine 5'triphosphate.
Incorporation of deoxy-6-thioguanosine 5'triphosphate into DNA has been
shown to trigger cell cycle arrest and apoptosis by a process that
involves the mismatch repair pathway (Swann et al., 1996). The
metabolism of 6-MP to TGMP is less direct than that of 6-TG, involving
two additional enzymes, inosine monophosphate dehydrogenase and
guanosine monophosphate synthetase. This difference is potentially
important as the first intermediate in this pathway, thioinosine
monophosphate can act as a substrate for TPMT, leading to the
production of S-methyl-thioinosine 5'-monophosphate
(MeTIMP), a strong inhibitor of purine de novo synthesis (PDNS) (Tay et al., 1969).
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It has been suggested that PDNS inhibition may make a significant
contribution to the cytotoxic action of 6-MP (Allan and Bennett, Jr.,
1971; Erb et al., 1998; Lancaster et al., 1998). We postulated that the
relative contribution to cell kill made by TGMP incorporation into DNA
and PDNS inhibition by MeTIMP may depend on the level of TPMT
expression. As the level of expression of TPMT varies greatly between
individual persons, partly because of genetic polymorphisms that
produce inactive forms of the enzyme (Szumlanski et al., 1996; Tai et
al., 1996; Krynetski et al., 1997; Otterness et al., 1997; De la
Moureyre et al., 1998; Spire-Vayron et al., 1999; Colombel et al.,
2000; McLeod et al., 2000; Seki et al., 2000), this has important
clinical significance, both for the selection of the agents to use in
clinical practice and in the propensity of the drugs to generate DNA
mutations (Relling et al., 1998, 1999; Thomsen et al., 1999; Pui and
Relling, 2000). To explore the effect of changes in TPMT expression on
the cytotoxic effect of 6-MP and 6-TG, we have transfected cDNA for the
human TPMT gene under the control of an inducible promoter
into human embryonic EcR293 cells and provide detailed evidence for the
role of TPMT in the modulation of thiopurine activity.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cell Lines and Transfection.
Human TPMT cDNA was subcloned
into the pIND vector of the ecdysone-inducible expression system
(Invitrogen, Paisley, UK) from a T84 clone kindly provided by Dr. R. Weinshilboum (Department of Pharmacology, Rochester, MN) (Szumlanski et
al., 1996). The linearized pIND vector was transfected into embryonic
kidney EcR293 cells pretransfected with the pVgRXR vector, as provided
by Invitrogen.
TPMT Assay.
TPMT activity in transfected cell lines was
determined in cell lysates using a radiochemical assay, as described
previously (Weinshilboum et al., 1978). Lysate protein concentration
was estimated using a commercially available kit (BCA Protein Assay; Perbio Science UK Ltd, Cheshire, UK) with bovine serum albumin as a
standard. Results were expressed as units per milligram of cellular
protein, where one unit is the amount of activity required to catalyze
the formation of 1 nmol of 6-methylmercaptopurine per hour.
Western Blotting.
Western blotting was performed using a
rabbit polyclonal antibody raised against human recombinant TPMT. Cell
pellets were re-suspended in 500 µl of ice-cold hypotonic buffer (10 mM Tris-HCl, pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride in
isopropanol and a protease inhibitor cocktail of
4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin A,
trans-epoxysuccinyl-L-leucylamideo(4-guanido)butane, bestatin, leupeptin, and aprotinin (Sigma, Poole, Dorset, UK) and
disrupted by sonication. Lysates were centrifuged at 7000 g for 5 min at 4°C, heated at 100°C for 5 min and stored at 
20°C before analysis.
using precast gradient gels [4-20%; Novex
(Invitrogen)] with 7.5 µg of protein per well. Enhanced chemiluminescence molecular weight markers (5 µl; Amersham
Biosciences, Little Chalfont, Buckinghamshire, UK) and 8 ng of
recombinant TPMT were run as control tracks.
Immunoblotting was performed using nitrocellulose membranes (0.45 µm
; Hybond-C; Amersham Biosciences). Nonfat dried milk (5%) in TBS/Tween
(0.1 M NaCl, 0.05% Tween 20, 0.01 M Tris-HCl, pH 7.5) was used to
block nonspecific binding. Membranes were exposed to TPMT primary
antibody (diluted 1000-fold in blocking buffer) for 1 h followed
by biotinylated swine anti-rabbit immunoglobulins (Dako, Ely,
Cambridgeshire, UK) (1/2000 in TBS/Tween) for 30 min and
Streptavidin-horseradish peroxidase (1/5000 in TBS/Tween) for 30 min.
Membranes were washed thoroughly in TBS/Tween between incubations.
Immune complexes were detected using an enhanced chemiluminescence kit
according to the manufacturer's instructions (Amersham Biosciences).
Measurement of Deoxythioguanosine Incorporation into DNA.
Deoxythioguanosine (dGs) incorporation into DNA
was measured using an adaptation of the method described by Warren et
al. (1995). DNA was extracted from 5 × 106
cells suspended in 200 µl of phosphate-buffered saline (PBS) using a
commercially-available spin column method according to the
manufacturer's instructions (QIAamp DNA Mini Kit; QIAGEN, Valencia,
CA). Purified DNA was eluted with 200 µl of 10 mM Tris-HCl, 0.1 mM
EDTA, pH 9.0. Before digestion and derivatization, DNA samples were
denatured at 100°C for 5 min followed by rapid chilling on ice. Ten
microliters of digestion buffer (500 mM sodium acetate buffer, 10 mM
MgCl2, pH 4.5) and 20 µl of 25 µg/ml
P1 nuclease (Roche Diagnostics Ltd, Lewes, UK) in
50 mM sodium acetate buffer, pH 4.5, containing 1 mM
MgCl2, were added to 100 µl of spin column eluent. After incubation at 42°C for 1 h, 20 µl of 1 M Tris,
pH 8.0, and 1 µl (1 unit) of calf intestinal alkaline phosphatase (Roche Diagnostics) were added and the mixture incubated at 37°C for
30 min before the addition of 10 µl of 400 mM formic acid and 60 µl
of methanol. Samples were derivatized overnight in the dark at room
temperature using 5 µl of a 1 mM solution of
N-[6-(7-amino-4 methylcoumarin-3-acetamido)
hexyl]-3'-(2'-pyridyldithio)propronamide (Pierce Chemical Company,
Rockford, IL) in dimethyl formamide.
, except that each run included an
isocratic phase from 0 to 10 min of 10% methanol, 90% 0.2 M formate
buffer, pH 4.0, for the separation of DNA nucleosides followed by a
linear gradient over 2 min to 40% methanol and 60% 0.2 M formate,
which was held isocratically for 25 min. Thymidine was detected by
absorbance at 461 nm. Compounds were identified on the basis of
retention time and quantified using calibration curves (Warren and
Slordal, 1993). Because dGs is unavailable
commercially, thioguanosine was used as a standard for this compound. A
10 mM solution of thioguanosine was made in 20 mM sodium hydroxide and
further dilutions were made in 10 mM Tris-HCl, 0.1 mM EDTA, pH 9.0. Thymidine standards were prepared in 10 mM Tris-HCl, 0.1 mM EDTA, pH
9.0. dGs incorporation into DNA was expressed as
the number of dGs residues per 100 thymidine
residues (dGs/100 T).
Measurement of Methylmetabolites of 6-Mercaptopurine and
6-Thioguanine.
Methylmetabolite standards were prepared as
follows. Stocks of 20 mM 6-MP, 6-TG, S-methyl mercaptopurine
(MeMP), S-methyl thioguanine (MeTG) and methylmercaptopurine
riboside (rMeMP) (Sigma) were prepared in 0.1 N
NaOH. MeTIMP and S-methyl thioguanosine monophosphate
(MeTGMP) were prepared from the incubation of freshly prepared
erythrocytes with rMeMP or MeTG, respectively.
Fresh blood collected into lithium heparin was centrifuged, the plasma
and buffy coat layer was removed, and the erythrocytes were washed
twice with PBS before resuspension at 25% (v/v) in 50 mM potassium
phosphate buffer, pH 7.4, containing 75 mM NaCl, 2 mM
MgSO4, 10 mM glucose, and 1 mM
rMeMP or MeTG. Suspensions were incubated for
22 h in an orbital shaker at 37°C. Perchloric acid (70%) was
added to a concentration of 10%, incubated on ice for 30 min, and then
centrifuged. The supernatant was removed and the pH adjusted to 8.0 with 10 M KOH. The sample was centrifuged to remove precipitated salt
and the supernatant concentrated approximately 15-fold by
centrifugation under vacuum. The concentrate was centrifuged at
14,000g for 10 min and 150-µl aliquots were separated by
HPLC using the separation procedure described by Krynetski et al.
(1995). Peaks corresponding to MeTIMP and MeTGMP were collected,
freeze-dried, and resuspended in 1 M Tris-HCl, pH 8.0. Confirmation
that the peaks collected were the correct compounds was shown by
heating MeTIMP and MeTGMP for 1 h at 100°C in 10% perchloric
acid, which gave a peak (by HPLC analysis) at the same retention time
as MeMP and MeTG treated in the same way, respectively. Removal of a
phosphate group from MeTGMP was also confirmed by treatment with
alkaline phosphatase resulting in a change in retention time by HPLC.
Treatment of MeTIMP with alkaline phosphatase produced a compound with
the same retention time as rMeMP. The correct
mass for MeTIMP and MeTGMP was confirmed by mass spectrum analysis
(data not shown).
. Cells
were washed twice in PBS after drug exposure and stored as pellets of
approximately 5 × 106 cells at 80°C.
For analysis, cells were resuspended in 500 µl of Tris-EDTA buffer
(10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and sonicated for 20 s. A
100-µl aliquot was removed for protein measurement as described
above. Remaining lysate was filtered using Centrikon-3K filters
(Millipore, Bedford, MA) in a microcentrifuge for 45 min at
20,800g and metabolites were measured in the filtrate.
Metabolite concentrations were expressed per milligram of protein.
Standards were diluted in the Tris-EDTA buffer and 500 µl of each was
added to a pellet of 5 × 106 untreated
cells and prepared as described for samples.
PDNS Assay.
The rate of PDNS was measured as described by
Masson et al. (1996). Cells were incubated for 2 h with
[14C]formate (Amersham Biosciences) to a final
specific activity of 127 d.p.m./pmol, concentration 0.5 mM. PDNS
was expressed as the amount of radiolabeled purine base (adenine and
guanine) relative to the respective nonradiolabeled base and the amount
of PDNS in experimental treatments calculated as a percentage of the
control cells.
Estimation of Doubling Times and Drug Sensitivity.
Cell
doubling times were determined by the sulforhodamine B (SRB) assay
(Skehan et al., 1990) and drug sensitivity was determined using both
the SRB and clonogenic assays. For the determination of drug
sensitivity, 96-well plates were seeded with 3 × 103 cells per well and TPMT expression was
induced using 3 µM MA for 24 h the day before drug or ethanol
(control vehicle) was added. Cells were incubated for 2.5 culture
doublings each. Percentage survival was calculated from the absorbance
measurements at 530 nm, using the no-drug control as 100%.
Statistical Methods. Drug sensitivity assays were analyzed using PRISM software (GraphPad, San Diego, CA) in which a sigmoidal dose-response curve (variable slope) or the spline LOWESS (point to point) curve analysis was fitted to all data for which drug concentrations were logged and the log IC50 measured from the curve automatically. Mean log IC50 values from separate experiments were compared by two-tailed unpaired t test and expressed as a mean + 95% CI in the original (un-logged) scale. Drug metabolite levels and PDNS measurements were compared, and hypotheses tested, by two-way ANOVA using Systat software (version 10.0; SPSS Inc., Chicago, IL).
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Characterization of TPMT Transfectants.
The clone showing the
highest level of induction, clone 20 (EcR293-TPMT/20), was selected for
further study. The optimum concentration required for TPMT induction
was determined by performing Western blotting for TPMT and TPMT
activity measurements after exposure for 24 h to 0 to 5 µM MA
(Fig. 2). Western blotting confirmed induction of TPMT protein. TPMT activity rose from a baseline of 0.69 U/mg of protein to reach a plateau of 2.67 U/mg. To determine the
stability of TPMT induction, EcR293-TPMT/20 cells were treated with 3 µM MA and TPMT activity was measured at 24, 48, and 72 h (Fig.
2C). There was a continued increase in TPMT activity after addition of
MA that began to reach a plateau after 72 h. The doubling time of
EcR293-TPMT/20 cells treated with MA was 29 h compared with
21 h for uninduced cells.
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The Effect of Increased TPMT Expression on Sensitivity to
Thiopurine Drugs.
To compensate for the lengthening of doubling
times after MA induction, cells were treated for an equivalent number
of cell doublings in both SRB and clonogenic assays. For the SRB
assays, IC50 values (the drug concentration
required to inhibit growth by 50%) were assessed from growth
inhibition curves. There was a small but statistically significant
decrease in sensitivity to 6-TG with IC50 values
rising on induction of TPMT from 1.74 µM (95% CI, 1.65 to 1.83 µM)
to 2.84 µM (95% CI, 2.25 to 3.59 µM, t4 = 3.73, P < 0.01) (Fig.
3A). Conversely, when the cells were
treated with 6-MP, there was an almost 4-fold increase in the
sensitivity to 6-MP in cells with induced TPMT with
IC50 values for the TPMT induced cells decreasing
to 1.78 µM (95% CI, 1.43 to 2.20 µM) from 8.01 µM for the
uninduced cells (95% CI, 3.30 to 19.42 µM, t5 = 5.34, P < 0.01) (Fig. 3B).
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The Effect of TPMT Expression on dGs Incorporation into
DNA.
An HPLC technique was used to determine whether alteration in
TPMT expression led to a change in levels of incorporation of dGs into DNA. Comparisons were made after 24-h
exposure to a range of concentrations of either 6-TG or 6-MP in cells
with or without induced TPMT (Fig. 5).
For both 6-TG and 6-MP there was a dose-dependent increase in
dGs incorporation into DNA.
dGs levels were significantly lower in TPMT
induced compared with uninduced cells using 6-TG at concentrations of
1.7 to 40 µM (two-way ANOVA on data for drug-treated cells, effect of
TPMT induction F1,16 = 25.578, P < 0.001) and 6-MP at concentrations of 1.8 to 80 µM (two-way ANOVA, effect of TPMT induction
F1,12 = 15.577, P < 0.01). At equitoxic concentrations of 6-TG (Fig. 5A) in TPMT-induced (2.8 µM 6-TG) and uninduced (1.7 µM 6-TG) cells there was no
significant difference in the incorporation of
dGs into DNA (hypothesis test,
F1,16 = 0.111, P > 0.7). In contrast, using equitoxic doses of 6-MP (Fig. 5B),
dGs levels were markedly lower in cells with
induced TPMT (1.8 µM 6-MP) compared with uninduced cells (8 µM
6-MP) (hypothesis test, F1,12 = 11.073, P < 0.01).
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Measurement of Levels of Methylated Thiopurine Metabolites.
The effect of TPMT expression on the extent of methylated thiopurine
metabolite production is shown in Fig. 6.
Cells were treated with 40, 4, 2.8, and 1.7 µM 6-TG or 80, 8, and 1.8 µM 6-MP, and levels of MeTG, MeMP, MeTIMP, and MeTGMP were measured after 24-h drug exposure. Irrespective of TPMT status, MeTG and MeMP
were not detected in cells exposed to 6-TG and 6-MP, respectively. With
40 µM 6-TG, increased TPMT expression led to a significant increase
in MeTGMP [0.25 ± 0.05 (S.D.) versus 0.04 ± 0.03 nmol/mg protein, t4 = 6.609, P < 0.01] (Fig. 6A). This metabolite was not detected in cells exposed to
lower concentrations of 6-TG or in cells exposed to 6-MP. With 80, 8, and 1.8 µM 6-MP, induction of TPMT led to a significant increase in
the level of MeTIMP (two-way ANOVA, effect of TPMT induction
F1,9 = 25.845, P < 0.001) (Fig. 6), but MeTIMP levels at equitoxic doses were similar
(two-way ANOVA, hypothesis test F1,9 = 0.542, p - 0.48).
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PDNS Measurements. At equitoxic doses of 6-MP, similar amounts of PDNS were observed (two-way ANOVA, F1, 18 = 0.02, P > 0.8 for adenine and F1,18 = 0.08, P > 0.7 for guanine; 1.8 µM 6-MP for TPMT induced versus 8 µM 6-MP for uninduced). However, for 6-TG, equitoxic doses produced a marked difference in PDNS, with TPMT-induced cells having higher PDNS than those not expressing TPMT (two-way ANOVA, F1,24 = 19.1, P < 0.001 for adenine and F1,24 = 20.67, P < 0.001 for guanine; 2.8 µM 6-TG for TPMT induced versus 1.7 µM 6-TG for uninduced).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The level of activity of the enzyme TPMT varies greatly between
individual subjects in part because of the presence of polymorphisms in
the TPMT gene that may affect either the enzymic activity or protein
stability (Szumlanski et al., 1996; Tai et al., 1996; Krynetski et al.,
1997; Otterness et al., 1997; De la Moureyre et al., 1998; Spire-Vayron
et al., 1999; Colombel et al., 2000; McLeod et al., 2000; Seki et al.,
2000). Approximately 10% of white persons carry a polymorphic
TPMT gene and biallelic polymorphisms occur in about 1 in
300 persons. (Otterness et al., 1997; Yates et al., 1997). Subjects
with this genotype have very low levels of TPMT activity; if they are
treated with standard doses of thiopurine drugs, they experience
profound myelosuppression. This is associated with greatly elevated
intracellular TGN levels, as determined in red blood cells.
By considerably reducing the dose used and carefully monitoring the
white blood cell count, it has been possible to reintroduce 6-MP in
patients with TPMT deficiency (Lennard et al., 1993; 1997a,b; Evans et
al., 1991; McLeod et al., 1993; Andersen et al., 1998). Interestingly,
it has been noted that the red blood cell TGN levels in these cases are
above the range seen in cases with normal TPMT activity (Erb et al.,
1998), suggesting that in the absence of TPMT, higher TGN levels are
required to produce equitoxic effects. One possible explanation for
this observation is that 6-MP exerts a cytotoxic effect by a mechanism
independent of TGN production, for example through inhibition of PDNS
by MeTIMP.
Treatment of transfected cells with 3 µM MA caused a 4-fold increase
in TPMT activity, comparable with the range we reported previously in
leukemic blasts (Coulthard et al., 1998).
The effect of enhanced TPMT expression on drug sensitivity, MeTIMP
production, DNA-TGN incorporation, and PDNS was found to differ
markedly for 6-MP and 6-TG. In the case of 6-TG, induction of TPMT led
to a 1.6-fold decrease in cytotoxic sensitivity as assessed by the SRB
assay. This was mirrored by a decrease in the level of DNA-TGN
incorporation. At equitoxic doses, the level of incorporation was
similar, supporting the hypothesis that for 6-TG, the chief mode of
cytotoxic action is incorporation of TGNs into DNA. MeTGMP, a weak
inhibitor of PDNS, was detectable only with 40 µM 6-TG but not at the
equitoxic doses. At equitoxic doses of 6-TG, there was a greater than
2-fold increase in the amount of PDNS in those cells with TPMT
induction. At equimolar doses, our results are comparable with those of
Dervieux et al. (2001), who showed that PDNS was greater when TPMT was
induced. These results, in combination with the finding of equivalent
amounts of DNA-TGNs at equitoxic doses, support the historical belief that the mechanism of cytotoxicity for 6-TG is dependent on DNA-TGN incorporation rather than inhibition of PDNS.
In marked contrast with the results obtained with 6-TG, induction of TPMT led to an increase in sensitivity to 6-MP of more than 4-fold as measured by the SRB assay. However, this was accompanied by a decrease in the level of DNA-TGN incorporation similar to that seen for 6-TG. At equitoxic doses of 6-MP, DNA-TGN levels fell to below the level of detection when TPMT expression was induced. After equimolar doses of 6-MP treatment, the level of MeTIMP showed a significant increase with a decrease in PDNS after TPMT induction.
However, at equitoxic doses of 6-MP in cells expressing high and low
TPMT, there were similar levels of PDNS and MeTIMP. Because MeTIMP is a
strong inhibitor of PDNS (Allan and Bennett, 1971), the fact that there
were similar levels of PDNS and MeTIMP at equitoxic doses suggests that
inhibition of PDNS is the main mechanism of cytotoxicity in these
cells. Reduced TGN incorporation at equitoxic doses is consistent with
diversion of 6-MP metabolism away from TGMP.
TPMT induction had no effect on the percentage of viable cells able to form colonies after exposure to 6-MP or 6-TG. In response to drug exposure, the total number of cells measured by SRB assay was affected by TPMT induction, with increase in cell number for cells treated with 6-TG and the converse for 6-MP. This would imply that TPMT alters the rate of proliferation without detectable effects on clonogenic ability at the doses used.
During the preparation of this article, Dervieux et al. (2001)
published an article reporting similar effects of TPMT transfection on
thiopurine sensitivity in the T-cell leukemic cell line CCRF-CEM; these
cells have been reported to have a defect in the MMR pathway (Taverna
et al., 2000), which may be expected to influence their sensitivity to
thiopurines, whereas the EcR293-TPMT/20 cells are MMR-proficient (data
not shown). A comparison of the level of TGN incorporation into DNA
after 24 h exposure to 6-TG indicates that for equivalent levels
of cytoxicity CCRF-CEM cells tolerate nearly 10 times the level of
DNA-TGN incorporation than MMR-proficient EcR293 cells. For transfected
CCRF-CEM (3.1 dGs/100 T) at 1 µM TG
(IC50 = 1.1 µM), 0.39 dG/100T at
IC50 for uninduced EcR293-TPMT/20 cells
(IC50 = 1.7 µM). These results indicate that the CCRF-CEM cells exhibit tolerance resistance to thiopurines, an
effect that may complicate the analysis of the results.
One important conclusion from our results is that, in cells with high
levels of TPMT, 6-MP can exert a cytotoxic effect without measurable
levels of TGN incorporation into DNA and, therefore, with reduced risk
of mutagenesis posed by TGN incorporation. This may help to explain
why, in contrast to 6-TG, secondary malignancy has not been associated
with chronic exposure to 6-MP or to the 6-MP prodrug azathioprine in
clinical practice. In cells with low TPMT, however, levels of TGN
incorporation in the DNA are much higher for a given level of
cytotoxicity. These results may provide a mechanistic explanation for
the recent observation that the rates of secondary malignancy are
raised in patients with congenitally reduced TPMT levels (Relling et
al., 1998, 1999; Thomsen et al., 1999; Pui and Relling, 2000). The
possible association between thiopurine treatment and mutagenic affects
needs further clarification to support these clinical findings. This is
currently being investigated using this model system. The results of
these experiments will help confirm if the lack of clinical benefit associated with 6-TG, plus the additional risk of mutagenesis and
secondary malignancy, would preclude the use of this drug in preference
to 6-MP.
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Acknowledgments |
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We thank Dr. A. Boddy for the mass spectrum analysis and Marian Case for proofreading this manuscript.
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Footnotes |
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Received November 9, 2001; Accepted March 25, 2002
This work was supported by grants from the Leukemia Research Fund and the North of England Children's Cancer Research Fund.
S.A.C. and L.A.H. contributed equally to the completion of this study. This work was previously presented in part in the Ph.D. dissertation of S.A.C., entitled "The role of thiopurine methyltransferase in the metabolism of cytotoxic drugs".
Address correspondence to: Dr. Andrew Hall, The LRF Molecular Pharmacology Specialist Programme, Cancer Research Unit, Medical School, Framlington Place, Newcastle Upon Tyne, NE2 4HH, UK. E-mail: a.g.hall{at}ncl.ac.uk
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Abbreviations |
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6-MP, 6-mercaptopurine; 6-TG, 6-thioguanine; ALL, acute lymphoblastic leukemia; TGN, thioguanine nucleotide; HGPRT, hypoxanthine guanine phosphoribosyltransferase; TPMT, thiopurine methyltransferase; TGMP, 6-thioguanosine 5'-monophosphate; MeTIMP, S-methyl-thioinosine 5'-monophosphate; PDNS, purine de novo synthesis; EcR, ecdysone receptor; MA, muristerone A; dGs, deoxythioguanosine; PBS, phosphate-buffered saline; HPLC, high-performance liquid chromatography; MeMP, S-methyl mercaptopurine; MeTG, S-methyl thioguanine; rMeMP, S-methyl mercaptopurine riboside; MeTGMP, S-methyl-thioguanosine 5'-monophosphate; SRB, sulforhodamine B; CI, confidence interval; ANOVA, analysis of variance; MMR, mismatch repair.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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relation to thiopurine metabolism.
Cancer
86:
1080-1086[CrossRef][Medline].This article has been cited by other articles:
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) or ethanol (CV) (
) treated for 24h with
various concentrations of either 6-TG (A) or 6-MP (B). Error bars
represent the S.E.M. of duplicates from three separate experiments.
