Measurement of cell kinetics in human tumours in vivo using bromodeoxyuridine incorporation and flow cytometry

Wilson, George D.; McNally, N. J.; Dische, S; Saunders, Michele I.; Rochers, C. Des; Lewis, Al; Bennett, Michael H

doi:10.1038/bjc.1988.234

Cited by 297 publications

(95 citation statements)

References 25 publications

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“…push over 2-4 min. Similar doses have been given in previous studies without report of toxicity (8,17,25,38), and no ill effects were noted in this study as well.…”

Section: In Situ Brdurd and Idurd Labeling In Patientssupporting

confidence: 88%

“…Thus, a shortening of overall treatment times might increase the chance of local control in properly selected patients. Recent studies (4)(5)(6)37,38) indicate a striking similarity between these effective tumor doubling times during treatment (14,18,19,21,39) and a quantity called the tumor potential doubling time, Tpot, measured prior to treatment. Tpot is the minimum time for tumor volume doubling, taking growth fraction into account but ignoring cell loss.…”

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confidence: 99%

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Tumor cell kinetics using two labels and flow cytometry

et al. 1994

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A flow cytometry based, single sampling method employing sequential bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd) labeling with an intervening interval is examined. BrdUrd-specific and nonspecific monoclonal antibodies and flow cytometry are used to estimate the duration of DNA synthesis and the potential doubling time in tumor cell populations using a single sampling or biopsy. A correction for labeled, divided cells allows postlabel incubation intervals to exceed the length of the G2/M phase of the cell cycle. The method yields reliable results when tested in experimental tumor systems in vitro and in situ, as well as in nine human tumors labeled in situ. It uses a somewhat simpler analysis than the alternative relative movement method, requiring only labeling indices, rather than both a labeling index and relative movement, but does require the administration of two, rather than one, label. It provides an independent verification of and a useful single sampling alternative to the relative movement method for the estimation of the length of S phase and, from this, the potential doubling time in experimental or clinical human tumors. 0 1994 Wiley-Liss, Inc.Key terms: Cell kinetics, bromodeoxyuridine, iododeoxyuridine, human tumors Analyses of both experimental and clinical data indicate decreased local tumor control with prolongation of overall radiation therapy treatment times (4,13, 18,31,39). Such an effect is likely to be due, at least in part, to tumor clonogenic proliferation during treatment. Thus, a shortening of overall treatment times might increase the chance of local control in properly selected patients. Recent studies (4-6,37,38) indicate a striking similarity between these effective tumor doubling times during treatment (14,18,19,21,39) and a quantity called the tumor potential doubling time, Tpot, measured prior to treatment. Tpot is the minimum time for tumor volume doubling, taking growth fraction into account but ignoring cell loss. These data indicate that many human tumors-about one-half of all head and neck tumors, for example (371-have the potential to double their cell number in as short a time as five or fewer days. Such considerations have stimulated much interest in the in situ measurement of rates of tumor cell proliferation.The delayed biopsy, relative movement technique (7) permits the estimation of Tpot from a single biopsy without the use of radioisotopes required by previous cell cycle analysis methods (31). It employs bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd) pulse labeling of the DNA of established tumor cell lines in vitro or of tumors in situ, followed, after a delay of several hours, by a single biopsy. Halogenated pyrimidine-specific monoclonal antibodies and propidium iodide staining of DNA are then employed with flow cytometry to determine the labeling index (LI) and a parameter called the relative movement that monitors the progression of labeled cells through the cell cycle. The length of S phase (T,) and the potential doubling time can then be ...

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“…push over 2-4 min. Similar doses have been given in previous studies without report of toxicity (8,17,25,38), and no ill effects were noted in this study as well.…”

Section: In Situ Brdurd and Idurd Labeling In Patientssupporting

confidence: 88%

mentioning

confidence: 99%

Tumor cell kinetics using two labels and flow cytometry

et al. 1994

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“…Proliferation can be studied by incorporation of the thymidine analogues bromo-and iododeoxyuridine, which can be identified using a monoclonal antibody with either flow cytometry analysis or immunohistochemical end points Wilson et al, 1985). Measurements of potential doubling time (Tpot) are currently being undertaken in a series of trials comparing conventional with accelerated fractionation, and some show good evidence that pretreatment cell kinetics can predict treatment outcome (Wilson et al, 1988;Begg et al, 1992; Corvo et al, 1995). The degree of hypoxia within a solid tumour is important as hypoxic cells will be up to three times more resistant to radiotherapy than their better oxygenated counterparts (Gray et al, 1953).…”

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Cell cycle distribution of hypoxia and progression of hypoxic tumour cells in vivo

Webster

Hodgkiss²,

Wilson³

1998

Br J Cancer

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Summary Hypoxia was assessed in three murine tumour models in vivo by measuring the incorporation of 7-(4'-(2-nitroimidazole-1-yl)-butyl)-theophylline (NITP), an immunologically identifiable hypoxia marker that binds bioreductively to cells under low-oxygen conditions. Proliferating cells were labelled in the same tumours by administering the thymidine analogue bromodeoxyuridine (BrdUrd). The relative hypoxia in each cell cycle phase of cells isolated from tumours was assessed by addition of propidium iodide with analysis by flow cytometry. There was no relationship between tumour volume and hypoxia in either the anaplastic sarcoma SaF or the poorly differentiated carcinoma CaNT and only a slight negative correlation in moderately well-differentiated carcinoma Rh. The G1/Go phase contained the greatest number of aneuploid hypoxic cells (aneuploid hypoxia ranging from less than 1% up to 40%, 38% and 71% in SaF, CaNT and Rh respectively), although there were significant amounts of hypoxia present in S-and G/M phases for all three tumours examined. However, the highest proportion of hypoxia occurred in the G/M phase, in which up to 60% of the cells were hypoxic. Simultaneous measurement of hypoxia, proliferation and DNA content using a novel triple-staining flow cytometry method showed that hypoxic cells could actively participate in the cell cycle. In addition, the cell cycle distribution of NITP and BrdUrd labelling showed that hypoxic cells could progress through the cell cycle, although their rate of progression was slower than that of better oxygenated cells.

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“…Measurements 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)(4)(5)12) or microscopic techniques (8,9).…”

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confidence: 99%

“…The amount of BrdUrd incorporated into DNA can be quantified by either flow cytometric (3)(4)(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.…”

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

et al. 1990

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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)

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Measurement of cell kinetics in human tumours in vivo using bromodeoxyuridine incorporation and flow cytometry

Cited by 297 publications

References 25 publications

Tumor cell kinetics using two labels and flow cytometry

Tumor cell kinetics using two labels and flow cytometry

Cell cycle distribution of hypoxia and progression of hypoxic tumour cells in vivo

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

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