Bromodeoxyuridine incorporation into DNA of human leukemia cells is not concentration dependent

Abstract

Leukemia blasts isolated from bone marrow aspirates of 44 adults with acute leukemia were incubated for 1 h with 0.008-32 pM bromodeoxyuridine (BrdUrd). After dual labeling with monoclonal anti-BrdUrd antibodies and propidium iodide, the cells were analyzed by flow cytometry. Percent labeled cells and intensity of labeling were similar over concentrations of BrdUrd ranging from 0.8-32 pM-a 40-fold range. Therefore, despite potential interpatient variability in nucleoside pharmacokinetics, commonly used doses of BrdUrd which are intended to achieve steady-state plasma concentrations in the 8.0 pM range can be expected to provide a reliable estimate of the S-phase fraction. Key terms: Cell kinetics, acute leukemia, concentration independence, flow cytometryMeasurements of incorporation of bromodeoxyuridine (BrdUrd) into DNA have provided a useful assessment of DNA synthesis in leukemia cells in vitro and in vivo (3)(4)(5)8,9,12). The amount of BrdUrd incorporated into DNA can be quantified by either flow cytometric (3-5,12) or microscopic techniques (8,9). If BrdUrd incorporation is to be a reliable in vivo measure, possible concentration-dependent artifacts must be avoided. Although interpatient variations in BrdUrd pharmacokinetics have not been extensively evaluated, continuous infusions of a fixed dose of the nucleoside cytosine arabinoside in leukemia patients can result in a 10-fold variation in plasma concentration a t steady state (1). It is expected that similar variations in plasma concentrations of other nucleosides such as BrdUrd might occur. BrdUrd doses (200 mgim' over 1 h) used in in vivo cytokinetic studies yield concentrations in the 8 pM range (5). A recent trial employed a lower dose of 100 mgim' (8); although pharmacokinetics were not evaluated in this study, elimination is expected to be linear (10). The toxicities of BrdUrd are dose dependent, but are not expected with the doses administered for cytokinetic studies (6).In an attempt to establish a range of concentrations over which BrdUrd incorporation into DNA of human leukemia cells is constant, the following experiment was performed. MATERIALS AND METHODSIsolation of Leukemia Cells A separate marrow aspirate from the posterior iliac crest of adult patients with acute leukemia was collected in heparinized syringes and resuspended in 30 cc of ice-cold RPMI medium 1640 (Gibco, Grand Island, NY). This suspension was layered over 15 ml of FicollPaque (Pharmacia, Picataway, NJ; density = 1.077) and centrifuged at 1,OOOg for 20 min. Cells at the plasma-Ficoll interface were harvested and washed twice with 50 ml of RPMI and then resuspended in 2 ml of RPMI with 10% fetal calf serum (FCS)

Telomerase activity in acute myelogenous leukaemia: clinical and biological implications

“…Cell cycle distribution was determined by staining DNA with propidium iodide (PI) (Sigma) as previously described ( Powell et al , 1990 ). To quantitate the amount of DNA per cell, 1 × 10 6 cells were incubated at 37°C for 1 h with 10 μ m of bromodeoxyuridine (BrdU) (Sigma).…”

Kinetic Heterogeneity of an Experimental Tumour Revealed by BrdUrd Incorporation and Mathematical Modelling

“…Some experimental evidence in vitro [Powell et al (1990) Starace and Santoro, unpublished results] shows that very low BrdUrd concentrations produce detectable labelling. After the BrdUrd injection in the animal, the label concentration in the fluids surrounding the tumour cells changes with the time and eventually vanishes.…”

Induction of apoptosis by vitamin D3 analogue EB1089 in NCI‐H929 myeloma cells via activation of caspase 3 and p38 MAP kinase

“…Cell cycle analysis Cell cycle distribution was determined by staining DNA with propidium iodide (PI; Sigma, St. Louis, MO, USA), as described previously ( Powell et al , 1990 ). To quantify the amount of DNA per cell, 1 × 10 6 cells were incubated with 10 µ m of bromodeoxyuridine (BrdU) (Sigma) at 37°C for 1 h. Cells were washed in PBS and fixed in 70% ethanol.…”