Dose-related effects of methotrexate on purine and pyrimidine nucleotides and on cell-kinetic parameters in molt-4 malignant human T-lymphoblasts

Bökkerink, J.P.M.; Abreu, Ronney A. De; Bakker, Marinka A.H.; Hulscher, Tilly W.; Baal, John M. van; Vaan, G. A. M. de

doi:10.1016/0006-2952(86)90626-x

Cited by 23 publications

(16 citation statements)

References 19 publications

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“…fall in binding from the mean Bo value. This is equivalent to a limit of detection of 0.4 pmol per 106 cells at an extract dilution of 1:5. dGTP pools in untreated cells measured by radioimmunoassay were similar to those reported for several mouse and human tumour cell lines measured by either high performance liquid chromatography (HPLC) (Bokkerink et al, 1986;Mattano et al, 1990) or DNA polymerase assays (Ross et al, 1981;Cohen et al, 1993). (Figure IA), CHO and HeLa cells dipyridamole completely blocked the rescue by hypoxanthine and the curves were the same as for lometrexol ± dipyridamole.…”

Section: Dgtp Radioimmunoassaysupporting

confidence: 80%

Selective potentiation of lometrexol growth inhibition by dipyridamole through cell-specific inhibition of hypoxanthine salvage

Turner

Aherne²,

Curtin³

1997

Br J Cancer

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Summary The novel antifolate lometrexol (5,10-dideazatetrahydrofolate) inhibits de novo purine biosynthesis, and co-incubation with hypoxanthine abolishes its cytotoxicity. The prevention of hypoxanthine rescue from an antipurine antifolate by the nucleoside transport inhibitor dipyridamole was investigated for the first time in nine human and rodent cell lines from seven different tissues of origin. In A549, HeLa and CHO cells, dipyridamole prevented hypoxanthine rescue and so growth was inhibited by the combination of lometrexol, dipyridamole and hypoxanthine, but in HT29, HCT116, KK47, MDA231, CCRF CEM and L1210 cells dipyridamole had no effect and the combination did not inhibit growth. Dipyridamole inhibited hypoxanthine uptake in A549 but not in CCRF CEM cells. Dipyridamole prevented the hypoxanthine-induced repletion of dGTP pools, depleted by lometrexol, in A549 but not in CCRF CEM cells. Thus, the selective growthinhibitory effect of the combination of lometrexol, dipyridamole and hypoxanthine is apparently due to the dipyridamole sensitivity (ds) or insensitivity (di) of hypoxanthine transport. Both the human and murine leukaemic cells are of the di phenotype. If this reflects the transport phenotype of normal bone marrow it would suggest that the combination of lometrexol, dipyridamole and hypoxanthine might be selectively toxic to certain tumour types and have reduced toxicity to the bone marrow.

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Section: Dgtp Radioimmunoassaysupporting

confidence: 80%

Selective potentiation of lometrexol growth inhibition by dipyridamole through cell-specific inhibition of hypoxanthine salvage

Turner

Aherne²,

Curtin³

1997

Br J Cancer

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“…MTX can exert its therapeutic effects by inhibition of DNA and RNA biosynthesis (4,18,23,30,36). Our studies in which Thd was applied concomitantly with MTX showed that the low dose of MTX used caused synchronisation through the inhibition of DNA biosynthesis (3,21,35). Compounds which specifically inhibit thymidylate synthase, such as N- (4- (N-((2-amino-4hydroxy-6-6-quinazolinyl) methyl) prop-2-ynyl amino) benzoyl-L-glutamic acid (221, might be even better than MTX for obtaining good synchronisation with minimal metabolic impairment.…”

Section: Discussionmentioning

confidence: 93%

Synchronisation of cancer cell lines of human origin using methotrexate

1990

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U937 human histiocytic lymphoma cell line and SW626 ovarian carcinoma line of human origin were synchronised using very low, nontoxic concentrations (0.04-0.08 pM for 16-24 h) of methotrexate (MTX) under standard culture conditions. Satisfactory synchrony was achieved to study S phase events. Various kinetic behaviours and biological properties of the synchronised cells are considered for characterisation of the system. MTX-synIn order to study the biological and biochemical events occurring at specific points of cell cycle progression, cells must be synchronised. Perfect synchronisation a t a specific point of the cell cycle is impossible without affecting the metabolic processes of the cells. Many methods have been used so far such as mitotic detachment, centrifugal elutriation, isoleucine deprivation, serum deprivation with hydroxyurea, double thymidine block, and sorting using flow sorting systems (1,19). None of them appear completely satisfactory either because the proportion of synchronised cells is not sufficiently high or because the cell manipulations perturb the cellular physiology too much. In addition, these methods are certainly not suitable for application in vivo. Thus, better methods need to be developed. Recently a method based on cell treatment with aphidicolin (APC), a reversible inhibitor of DNApolymerase a, has been proposed and successfully used to investigate cells of different stages of progression in S phase (33). Although this method achieves a high degree of synchronisation, the concentration of APC can cause significant cytotoxicity (10). Prolonged exposure to low doses of methotrexate (MTX) has also been proposed for synchronisation (37) and has been used by cytogeneticists for high resolution banding analysis. The method, however, has not yet been sufficiently characterised. We describe the synchronisation of two cancer cell lines of human origin in culture using low doses of the antifolate, MTX. The aim was to achieve satisfactory synchrony to study S-phase events. The chronisation was compared with that induced by aphidicolin (APC) alone and by serum deprivation and APC. In some cancer cell lines MTX appears to be the best choice for obtaining highly synchronised cell populations without cytotoxicity or physiological perturbations. Key terms: BrdUrd, flow cytometry, aphidicolinvarious kinetic behaviours and biological properties of the synchronised cells during reversal of the process are also considered for better characterisation of the system. MATERIALS AND METHODSReagents MTX was obtained from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, MD. Aphidicolin-17-glycinate HC1 was kindly provided by ICI, UK. Thymidine (Thd), anti-mouse IgG conjugated with FITC developed in goat were from Sigma Chemical Co. (St. Louis, MO). Bromodeoxyuridine (BrdUrd) and antiBrdUrd were products of Becton Dickinson (Mountain View, CAI. Hypoxanthin (HX) was a product of E. Merck (FRG). Propidium iodide (PI) and normal goat serum were purch...

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“…MTX can inhibit DNA and RNA biosynthesis [20][21][22][23][24] and the effect is dependent on the intracellular accumulation of the polyglutamated form of the drug [25]. Our studies [1] in which thymidine was concomitantly applied with MTX showed that the low dose of MTX caused cell synchronization by inhibition of DNA biosynthesis [26][27][28]. Compounds that specifically inhibit DNA synthesis, such as CB3717 [29], migh produce better results than MTX for inducing synchronization.…”

Section: Discussionmentioning

confidence: 99%

Synchronization of cancer cell lines with methotrexate in vitro

Erba

Şen

1996

Methods Cell Sci

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Detailed procedure are described for inducing synchrony in cancer cell lines of human origin (Jurkat, K562, U937, SW626) and L1210 cell line of murine origin by using very low non-toxic concentrations (0.04-0.08 gM for 13-24 hours) of methotrexate under standard culture conditions. This protocol offers a method for synchronization of cells at the G~/S boundary and through the S-G2-M phases of the cell cycle. Kinetic behavior and biological

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Dose-related effects of methotrexate on purine and pyrimidine nucleotides and on cell-kinetic parameters in molt-4 malignant human T-lymphoblasts

Cited by 23 publications

References 19 publications

Selective potentiation of lometrexol growth inhibition by dipyridamole through cell-specific inhibition of hypoxanthine salvage

Selective potentiation of lometrexol growth inhibition by dipyridamole through cell-specific inhibition of hypoxanthine salvage

Synchronisation of cancer cell lines of human origin using methotrexate

Synchronization of cancer cell lines with methotrexate in vitro

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