Synchronisation of cancer cell lines of human origin using methotrexate

Abstract

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...

DNA fragmentation, dATP pool elevation and potentiation of antifolate cytotoxicity in L1210 cells by hypoxanthine

“…However, measurements of RNA and DNA contents of CCRF-CEM (F2) cells treated with 0.1 mM MTX have shown that while HX allowed continued RNA synthesis, the block in cell cycle progression was only overcome by the addition of thymidine and HX (Taylor & Tattersall, 1981). This inability of HX to alleviate the block was also demonstrated in other cell lines (Sen et al, 1990).…”

Mechanism of cytotoxicity of 5,10-dideazatetrahydrofolic acid in human ovarian carcinoma cells in vitro and modulation of the drug activity by folic or folinic acid

“…Cells and culture conditions The SW626 human ovarian carcinoma cell line was used (Sen et al, 1990). For all these experiments cells were grown as monolayers in RPMI-1640 medium supplemented with 10% dialysed fetal bovine serum (FBS) (cut-off point 3,500 Da).…”

“…For biparametric BrdU/DNA analysis (Sen et al, 1990), 30 JLM BrdU was added to the cells for 30 min at different times during DDATHF treatment and after drug washout, and fixed with 70% ethanol at 4°C. The cells were washed with phosphate-buffered saline (PBS) and DNA was denatured with 3 M hydrochloric acid for 30 min at room temperature.…”

Sorting of cells from different cell cycle phases using surface antigen expression

“…The aim of this methodology is to provide a technique able to separate cells in different cycle phases avoiding the use of specific drugs such as methotrexate or aphidicolin [1,2]. Due to our experience in working with physical parameters in order to discriminate nuclear subsets we have applied a multiparametric approach based on scattering properties of cells and expression of surface antigens.…”