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Abstract
The expression of thymidine kinase--an enzyme of the DNA precursor pathway--is strictly regulated during the normal cellular cycle, but is much higher and permanently expressed in malignant growing cells. We used this fact to detect neoplastic cells in samples freshly taken from leukaemia patients and kept frozen in liquid nitrogen until analysis. Using a new cytofluorometric assay for thymidine kinase in single cells, we were able to identify leukaemic cells in a surplus of normal ones. Our results demonstrate the benefits of this assay for leukaemia diagnosis.
Highlights
The activity of Thymidine kinase (TK) is strictly regulated during the normal cell cycle, peaking at the onset of DNA synthesis but remaining extremely low in resting cells (Bello, 1974; Wawra et al, 1981)
In order to determine the amount of DNA in the presence of DAUdR, living cells had to be stained with ethidium bromide
Growing cells have low TK activity in G, this activity increases during early S-phase and returns to about the original level in G2
Summary
Samples from patients suffering from acute lymphocytic leukaemia (ALL) or acute myelogeneous leukaemia (AML) were taken at the time of diagnosis. After washing three times in phosphate-buffered saline (PBS), the leucocytes were resuspended in RPMI-1640 medium containing 20% human AB plasma and 10% dimethylsulphoxide. Buffy coat, obtained by leukapheresis, stimulated lymphocytes, and bone marrow samples from patients were treated and frozen as described above. Non-stimulated leucocytes from leukaemia patients and normal controls, phytohaemagglutinin-stimulated normal lymphocytes and bone marrow cells were all thawed as fast as possible in a 37°C water bath and transferred into 5 ml of RPMI-1640 medium containing 20% fetal calf serum. After 30 min, cells were washed with RPMI-1640 medium without serum and incubated with the fluorescent thymidine analogue as described below. DNA staining was performed with 1.5 pg ml-' ethidium bromide for 10-20 min on ice. Cells were analysed within the following 20 min to ensure that they were still alive and that the phosphorylated thymidine analogue was not washed out. Dyes; filters were set to obtain a complete separation of emission of the fluorescent analogue (500 nm) and of ethidium bromide (605 nm)
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