Cytofluorometric determination of thymidine kinase activity in a mixture of normal and neoplastic cells

Abstract

A cytofluorometric assay allowing the measurement of thymidine phosphorylation in single cells had been established (Hengstschläger & Wawra, 1993). This assay enables us to correlate intracellular thymidine kinase (TK) activity with the DNA content of single cells. Enzyme activity levels from neuroblastoma cells and normal fibroblasts derived from the same patient were determined. Using this cytofluorometric assay in a mixture of both cell types the neoplastic cells could be distinguished from the normal fibroblasts because of their higher TK level. A human lymphoblastoid cell line was compared with the cell line KG-1, derived from an acute myelogenous leukaemia, in the same way. The increased enzyme activity enabled us to detect KG-1 cells in a mixture with an 10,000-fold excess of Epstein Barr virus transformed lymphocytes.

Identification of leukaemic cells in bone marrow and blood samples by a new cytofluorometric assay

“…In an earlier report, we analysed artificial mixtures of growing normal and malignant cells and were able to identify tumour cells mixed with a 10 000-fold excess of normal ones (Hengstschlager and Wawra, 1993b). From the data in the present study, we may conclude that a total of at least some 50 or 100 malignant cells are necessary for identification under average conditions.…”

“…In the past, we have established a cytofluorometric method based on the uptake of a fluorescent nucleoside that is subsequently phosphorylated by the cells in the same manner as thymidine (Hengstschlager and Wawra, 1993a (Hengstschlager et al, 1994b). Moreover, this method enabled us to identify a few transformed cells mixed with a 10 000-fold excess of normal ones (Hengstschlager and Wawra, 1993b isolated from heparinised peripheral blood of leukaemia patients and from two normal subjects using the FicollHypaque gradient method. 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.…”

“…When applied to cells in culture, our new cytofluorometric assay allows us to discriminate between normal growing cells, like diploid fibroblasts, and virally transformed cells or lines derived from tumours (Hengstschlager et al, 1994b). Moreover, this method enabled us to identify a few transformed cells mixed with a 10 000-fold excess of normal ones (Hengstschlager and Wawra, 1993b ethanol) at 37°C and 7.5% carbon dioxide. After harvesting by centrifugation for 5 min at 1000 r.p.m., cells were washed twice with PBS.…”

Expression of the cyclin‐dependent kinase inhibitor p16 during the ongoing cell cycle

“…Protein extracts were prepared as described [19]. After washing with phosphate-buffered saline cell lysis was performed using a buffer consisting of 10 mM Tris-HCl (pH 7.5), 250 mM sucrose, 160 mM KC1, 1.5 mM MgC12, 3 mM fl-mercaptoethanol, 50 mM e-amino-n-capronic acid, and 0.8 mg/ml digitonin.…”

Fetal cells in the peripheral blood of pregnant women express thymidine kinase: a new marker for detection

“…Synthesis and purification of the fluorescent thymidine analogue AUdR/DANS were published previously, and cytofluorometric measurement of TK activity in living cells was performed as described [11,12]. Cells were exposed in RPMI medium without serum for 30 min to 1.5 mM AUdR/DANS (a stock solution was prepared in 70% ethanol) at 37°C and 7.5% C0 2 .…”