Effects of methotrexate on purine and pyrimidine metabolism and cell-kinetic parameters in human malignant lymphoblasts of different lineages

Bökkerink, J.P.M.; Abreu, Ronney A. De; Bakker, Marinka A.H.; Hulscher, Tilly W.; Baal, John M. van; Schretlen, E. D. A. M.; Bruijn, Chris H. M. M. de

doi:10.1016/0006-2952(88)90359-0

Cited by 31 publications

(16 citation statements)

References 37 publications

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“…6,7 The consequence of DNPS inhibition by these antimetabolites is purine deprivation, leading to inhibition of DNA synthesis, decreased cell proliferation, and cytotoxicity. 8 Although in vitro studies have demonstrated that DNPS inhibition by MTX and/or MP contributes to their cytotoxic effects 6,8 and that MTX putatively acts synergistically with MP by enhancing the accumulation of PRPP, a necessary cofactor for MP activation, 9,10 these mechanisms have not been established in primary leukemia cells in vivo.…”

Section: Introductionmentioning

confidence: 99%

De novo purine synthesis inhibition and antileukemic effects of mercaptopurine alone or in combination with methotrexate in vivo

Dervieux

Brenner

Hon

et al. 2002

Blood

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Methotrexate (MTX) and mercaptopurine (MP) are widely used antileukemic agents that inhibit de novo purine synthesis (DNPS) as a mechanism of their antileukemic effects. To elucidate pharmacodynamic differences among children with acute lymphoblastic leukemia (ALL), DNPS was measured in leukemic blasts from newly diagnosed patients before and after therapy with these agents. Patients were randomized to receive lowdose MTX (LDMTX: 6 oral doses of 30 mg/m 2 ) or high-dose MTX (HDMTX: intravenous 1 g/m 2 ) followed by intravenous MP; or intravenous MP alone (1 g/m 2 ), as initial therapy. At diagnosis, the rate of DNPS in bone marrow leukemia cells was 3-fold higher in patients with T-lineage ALL compared with those with B-lineage ALL (769 ؎ 189 vs 250 ؎ 38 fmol/nmol/h; P ‫؍‬ .001). DNPS was not consistently inhibited following MP alone but was markedly inhibited following MTX plus MP (median decrease 3% vs 94%; P < .001). LDMTX plus MP and HDMTX plus MP produced greater antileukemic effects (percentage decrease in circulating leukocyte counts) compared with MP alone (؊50% ؎ 4%, ؊56% ؎ 3%, and ؊ 20% ؎ 4%, respectively; P < .0001). Full DNPS inhibition was associated with greater antileukemic effects compared with partial or no inhibition (؊63% ؎ 4% vs ؊37% ؎ 4%; P < .0001) in patients with nonhyperdiploid B-lineage and T-lineage ALL.HDMTX plus MP yielded 2-fold higher MTX polyglutamate concentrations than LDMTX plus MP (2148 ؎ 298 vs 1075 ؎ 114 pmol/ 10 9 cells; P < .01) and a higher percentage of patients with full DNPS inhibition (78% vs 53%; P < .001). Thus, the extent of DNPS inhibition was related to in vivo antileukemic effects, and a single dose of intravenous MP produced minimal DNPS inhibition and antileukemic effects, whereas MTX plus MP produced greater antileukemic effects and DNPS inhibition, with full inhibition more frequent after

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Section: Introductionmentioning

confidence: 99%

De novo purine synthesis inhibition and antileukemic effects of mercaptopurine alone or in combination with methotrexate in vivo

Dervieux

Brenner

Hon

et al. 2002

Blood

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“…In studies of high-dose MTX therapy, Bokkerink et al 23,24 showed the time-and concentration-dependent effects of MTX on malignant human lymphoblasts. They also demonstrated that exposure to 0.02 µM MTX could inhibit DNA synthesis.…”

Section: Discussionmentioning

confidence: 99%

Pharmacokinetics and efficacy of low-dose methotrexate in patients with rheumatoid arthritis

et al. 2004

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This article evaluates the relationship between the pharmacokinetics of methotrexate (MTX), its efficacy in the treatment of rheumatoid arthritis (RA), and serum folic acid (FA) levels. The pharmacokinetics of MTX was studied in 29 patients with RA treated with low-dose MTX. The weekly dose of MTX was given orally at 2-4 mg every 12 h over a period of 24-36 h. Blood samples were taken 4 h after the first administration in any given week. A Bayesian method was used to estimate individual MTX pharmacokinetic variables. We then investigated the efficacy of MTX and the serum FA levels in these patients. We examined C-reactive protein levels (CRP) and the erythrocyte sedimentation rate (ESR), and analyzed the values obtained before and after MTX treatment in order to evaluate the efficacy of the MTX treatment. The degree of improvement in CRP and ESR was significantly correlated with the length of time the MTX concentration-time curve remained above 0.02 microM in one week. Furthermore, the degree of improvement in CRP was also significantly correlated with the area under the concentration-time curve (AUC) for MTX. These results suggest that serum MTX measurements could be useful in determining individual patient regimens.

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“…Depletion of TTP pools is one of the early biochemical events induced by MTX treatment (Bokkerink et al, 1988). Such dNTP imbalance has been reported to induce DNA strand breaks, presumably through an activation of endonuclease (Yoshioka et al, 1987).…”

Section: Changes In 7tp Pools Following Mtx Treatmentmentioning

confidence: 99%

“…Consequently, cells are depleted of both thymidylate and purine nucleotides (Bokkerink et al, 1988), since tetrahydrofolates act as co-factors in their biosynthesis. Therefore, the growth-inhibitory effects of MTX can be explained by thymine-less and/or purine-less mechanisms.…”

mentioning

confidence: 99%

“…Methotrexate (MTX) limits the intracellular supply of reduced folates (tetrahydrofolate) through inhibition of dihydrofolate reductase (DHFR) (Werkheiser, 1963;Jolivet et al, 1983). Consequently, cells are depleted of both thymidylate and purine nucleotides (Bokkerink et al, 1988), since tetrahydrofolates act as co-factors in their biosynthesis. Therefore, the growth-inhibitory effects of MTX can be explained by thymine-less and/or purine-less mechanisms.…”

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confidence: 99%

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Increased methotrexate‐induced DNA strand breaks and cytotoxicity following mutational loss of thymidine kinase

Sano

Kubota

Kasai

et al. 1991

Intl Journal of Cancer

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M T X for 16 hr, TK-cells showed a significantly higher number of DNA strand breaks. Concomitahtly, DNA fragmentation at the nucleosomal linker region was detected more prominently in TK-cells. Although MTX tended to decrease l T P pools similarly in all 3 cell types, the initial l T P level in TK-cells was only about one-fifth of that found in the wild type. These results indicate that the thymidine salvage pathway has a pivotal role in mediating MTX-induced toxicity and DNA lesions.Methotrexate (MTX) limits the intracellular supply of reduced folates (tetrahydrofolate) through inhibition of dihydrofolate reductase (DHFR) (Werkheiser, 1963;Jolivet et al., 1983). Consequently, cells are depleted of both thymidylate and purine nucleotides (Bokkerink et al., 1988), since tetrahydrofolates act as co-factors in their biosynthesis. Therefore, the growth-inhibitory effects of MTX can be explained by thymine-less and/or purine-less mechanisms. In fact, MTXinduced cytotoxicity is reversed by the simultaneous addition of thymidine and purine bases such as hypoxanthine at appropriate concentrations (Hakala and Taylor, 1969). However, there is some controversy as to whether the thymine-less or the purine-less state is more relevant to the cytotoxicity.The accumulation of DNA strand breaks following MTX treatment has been reported in Ehrlich ascites tumor cells (Li and Kaminskas, 1984). Several groups have confirmed this observation in other cell lines, such as human colon adenocarcinorna cells (Lonn and Lonn, 1988) and NIH/3T3 cells (Lorico et al., 1988). Although the biochemical mechanism remains to be elucidated, one attractive hypothesis involves the activation of endonuclease through imbalances in deoxyribonucleoside triphosphate (dNTP) pools (Yoshioka et al., 1987). Similar DNA lesions had been induced during treatment with 5-fluorodeoxyuridine (Yoshioka et al., 1987), which inhibits thymidylate synthetase following phosphorylation into its mononucleotide.This report presents the results of biochemical genetic studies of the role of a thymidine or purine salvage pathway in MTX-induced DNA strand breaks. MATERIAL AND METHODS Cell linesA human promyelocytic leukemia cell line, HL-60, came from the Japanese Cancer Resources Bank (Tokyo, Japan). Cells were grown in suspension culture in RPMI-I640 medium supplemented with 10% heat-inactivated dialyzed fetal bovine serum, 2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 p g h l ) . Dialysis of the serum is necessary to minimize the influence of several nucleosides or nucleobases contained in the fetal bovine serum. The cultures were maintained at 37°C in a humidified atmosphere of 95% air/5% CO,. HGPRT-deficient (HGPRT-) or TK-deficient (TK-) HL-60 cells were selected in our laboratory on the basis of their resistance to either 8-azaguanine or bromodeoxyuridine after mutagenesis with ethylmethane sulfonate (Kubota et al., 1983). The drug-resistant population was cloned by limiting dilution (0.5 cell/well) in 96-well tissue culture trays. The clones analyzed in...

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Effects of methotrexate on purine and pyrimidine metabolism and cell-kinetic parameters in human malignant lymphoblasts of different lineages

Cited by 31 publications

References 37 publications

De novo purine synthesis inhibition and antileukemic effects of mercaptopurine alone or in combination with methotrexate in vivo

De novo purine synthesis inhibition and antileukemic effects of mercaptopurine alone or in combination with methotrexate in vivo

Pharmacokinetics and efficacy of low-dose methotrexate in patients with rheumatoid arthritis

Increased methotrexate‐induced DNA strand breaks and cytotoxicity following mutational loss of thymidine kinase

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