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Drug Monitoring and Toxicology |
a Author for correspondence. Fax (+)31-24-3616428; e-mail r.deabreu{at}ckslkn.azn.nl.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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6-MP and its metabolites can be methylated by thiopurine methyltransferase (TPMT). The methyl donor is S-adenosyl-L-methionine (AdoMet), which is formed from adenosine triphosphate (ATP) and methionine.
We have described previously that the methylation of thiopurines leads to depletion of AdoMet (7)(8)(9). Furthermore, methyl-tIMP is an inhibitor of de novo purine synthesis; as a consequence, ATP becomes depleted and conversion of methionine to AdoMet may be hampered (9).
AdoMet is a universal methyl donor and is involved in the methylation of several molecules, e.g., DNA, RNA, and proteins. Depletion of AdoMet could lead to an altered methylation state and function of DNA and RNA (10). DNA methylation can be important for the expression of genes. In general, methylation of the promoter region of a gene is associated with a block of transcription (11).
Several studies have shown a genetic polymorphism for TPMT, with high- and low-activity alleles having been demonstrated (12)(13). This wide interindividual variation leads to the conclusion that patients with a low TPMT activity have lesser quantities of methylated thiopurines. These patients can have an increased DNA methylation, which might result in altered gene expression. However, further investigation is needed to tell more about the relation between TPMT activity and DNA methylation.
In the present study, we determined whether exposure of leukemic cells to 6-MP affected the methylation state of newly synthesized DNA. We used the MOLT F4 cell line (a human malignant lymphoblastic T-cell line) as a model.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cell culture
The experiments were performed with MOLT F4. The cells were
cultured in RPMI 1640 medium supplemented with 100 mL/L nondialyzed
fetal calf serum, 2 mmol/L L-glutamine, 2 mmol/L
sodium pyruvate, and 50 mg/L gentamicin. The culture medium was
refreshed thrice a week. The cells were maintained in a 50 mL/L
CO2 humidified atmosphere at 37 °C. The absence of
mycoplasma contamination and the presence of marker antigens were
tested regularly.
procedures
Treatment with 6-MP followed by pulse-labeling.
The
cells were kept in a logarithmic phase before drug exposure. The
experiments started at an initial cell number of 0.3 x
10 cells/mL. After 24-h exposure to 6-MP concentrations of
0.5, 1, 2, and 10 µmol/L, the cells were transferred to a 6-well
plate and radioactive pulse label was added. The final volume was 500
µL. For pulse labeling we added 1.5 mmol/L
L-[methyl-C]methionine (DuPont NEN; 44
Ci/mol) as methyl donor and 0.2 µmol/L
[methyl-H]thymidine (Amersham; 52 kCi/mol) to measure
newly synthesized DNA. After 4-h incubation at 37 °C in the presence
of the pulse labels, the cells were harvested for DNA isolation
according to the method of Miller et al. (14).
Measurement of the incorporated radioactivity.
All
experiments were performed in duplicate. In experiments 1–3, a small
amount of the DNA solution was transferred into a counting vial and the
amount of incorporated radioactivity was measured in a liquid
scintillation counter. In experiment 4, to demonstrate that the
methylation of deoxycytidine residues was reduced, we performed an
enzymatic hydrolysis of the isolated DNA as described below. The
hydrolyzed samples were then injected into the HPLC and the
5-methyldeoxycytidine and deoxythymidine fractions were collected.
Radioactivity of the collected fractions was measured in a liquid
scintillation counter.
DNA hydrolysis.
The isolated DNA was hydrolyzed
according to the method of Gehrke et al. (15). In short,
DNA was treated with nuclease P1 and bacterial alkaline
phosphatase (type III) to obtain nucleosides that could be separated
and detected by HPLC. The hydrolyzed DNA was stored at -20 °C until
HPLC analysis.
HPLC analysis of hydrolyzed DNA.
The HPLC system
consisted of an isocratic pump (Thermo Separation Products) and a
Supelcosil LC-18S column (25 cm x 4.6 mm; 5 µm particle size;
Supelco). The eluent, 50 mmol/L K2HPO4 (pH 4.0)
with 20 mL/L methanol, was delivered to the column at a flow rate of 1
mL/min. The detector (Separations; Model 759A) was set at 285 nm
(
max of 5-methyldeoxycytidine).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, experiments 1–3).
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Treatment with 0.5 µmol/L 6-MP had very little effect on the C/H ratio, compared with the untreated cells (P >0.05). However, the C/H ratio was decreased by 17% with 1 µmol/L 6-MP, by 50% with 2 µmol/L 6-MP, and by 70% with 10 µmol/L 6-MP.
Radioactivity of methyldeoxycytidine and deoxythymidine was measured
more specifically after DNA hydrolysis and HPLC analysis (Fig. 1
). A similar decrease of the C/H
ratio was found by this method (Table 1
, experiment 4). The validation
of the HPLC method was tested by measuring the
C/H ratios of several dilutions of DNA from
untreated cells and from cells treated with 6-MP. In these validation
experiments we compared the results found by direct liquid
scintillation counting with results found after enzymatic hydrolysis,
HPLC, and liquid scintillation counting of the methyldeoxycytidine and
deoxythymidine fractions. The C/H ratios of
both procedures were similar: 0.22 ± 0.01 and 0.24 ± 0.02
for the untreated cells and 0.13 ± 0.01 and 0.14 ± 0.01 for
the 6-MP-treated cells, respectively.
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Formation of newly synthesized DNA was not affected by the treatment with increasing concentrations of 6-MP. The incorporated H was in the same range in the treated cells as in the untreated cells (with the SD of 10%).
An example of a chromatogram for the HPLC analysis of hydrolyzed DNA is
given in Fig. 1
. Methyldeoxycytidine and deoxythymidine were well
separated.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We also determined (10) that, concomitantly, AdoMet was depleted and S-adenosyl-L-homocysteine (AdoHcy) and methionine were increased. The AdoMet concentration and the AdoMet/AdoHcy ratio are indications for the methylation capacity. The AdoMet/AdoHcy ratio decreased from 4.5 in untreated cells to 1.1 in cells treated with 10 µmol/L 6-MP. We suggested that under these conditions a reduced methylation of DNA may occur.
Ordinarily, DNA methylation patterns are preserved to the next generations of cells. Synthesis and methylation are closely coupled processes (13), and a reduced AdoMet concentration and a decreased AdoMet/AdoHcy ratio can interfere with these processes and lead to a reduced DNA methylation.
To investigate the effect of 6-MP on DNA methylation, we performed experiments in which we treated MOLT F4 lymphoblasts with various concentrations of 6-MP. The concentrations used were in the same range as measured in vivo during high-dose 6-MP therapy (15).
Incubation with L-[methyl-C]methionine leads to formation of [methyl-C]-AdoMet, which is used as methyl donor by DNA methyltransferase. As a result, [methyl-C] will be incorporated into DNA. The C incorporation in DNA is a measure for the DNA methylation.
The synthesis of newly formed DNA is determined by the incorporation of H (derived from [methyl-H]thymidine) into DNA. Methylation of newly formed DNA is indicated by the C/H ratio. Treatment with increasing concentrations of 6-MP resulted in a decrease of the C/H ratio. This, together with the fact that the formation of newly synthesized DNA was not affected under above conditions, led us to conclude that the decrease of the C/H ratio is caused by a reduced incorporation of C in DNA. Therefore, hypomethylation will occur during incubation with 6-MP.
As indicated previously (9), the concentration-dependent decrease of methylation resembled the reduction of the ATP concentration with increasing concentrations of 6-MP. These effects on the DNA methylation were observed only with 6-MP concentrations >1 µmol/L.
The results of experiment 4 (Table 1
), where we specifically measured
the incorporation of [methyl-C]deoxycytidine and
[H]deoxythymidine into DNA, resemble the results of the
first three experiments. That is, the methylation of deoxycytidine
residues in DNA is reduced and hypomethylation occurs. This
hypomethylation may have a great impact: New restriction sites may be
created and even the expression of genes can be influenced. As pointed
out, 6-MP can reverse DNA methylation and may act in two ways: First,
expression of a tumor suppressor gene may be enhanced and in this way
may block oncogenesis. On the other hand, 6-MP may induce
hypomethylation of protogenes and may activate these genes to
oncogenes; in this way, 6-MP may induce carcinogenesis. However,
possible promoting effects of 6-MP on carcinogenesis have been studied
recently in rats by Matsushima et al. (17), who found no
significant influence of 6-MP on carcinogenesis. Further experiments
are needed to investigate the specific effect of hypomethylation by
6-MP on gene expression.
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Acknowledgments |
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Footnotes |
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1 Nonstandard abbreviations: 6-MP, 6-mercaptopurine; tIMP,
thio-IMP; TPMT, thiopurine methyltransferase; AdoMet,
S-adenosylmethionine; and AdoHcy,
S-adenosylhomocysteine. 
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References |
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