An improved mathematical method to estimate DNA synthesis time of bromodeoxyuridine-labelled cells, using FCM-derived data

Abstract. Chinese hamster ovary cells in vitro were pulse-labelled with bromodeoxyuridine (BrdUrd and were then allowed to progress through the cell cycle. Every half hour after labelling, cells were harvested and prepared for simultaneous flow cytometric determination of DNA content and incorporated BrdUrd, with the intercalating dye propidium iodide and with a monoclonal antibody against incorporated BrdUrd, respectively. The relative movement (RM), i.e. the relative mean DNA content of the moving cohort of BrdUrd-labelled cells in relation to that of G₁ and G₂ cells, was calculated. RM was then used to calculate DNA synthesis time (T_S), at all post-labelling times (t). Since labelled cells in G₂ and mitosis (M) in addition to S phase cells, are included in the cohort of moving labelled cells, and since the time of G₂ and M (T_g2+M) phases is finite, a non-linear relationship exists between RM and post-labelling time. Because of this, the use of a linear formula in the calculation of T_S yields results that are affected by t. We found that RM data can be corrected with regard to T_G2+M resulting in the derivation of a non-linear T_S formula. This non-linear T_S formula gave results that were nearly independent of t. Moreover, windows were set in the mid DNA distributions for G₁, S and G₂+ M cells in the bivariate DNA v. BrdUrd cytograms, to estimate the fraction of BrdUrd-labelled cells in each window at every post-labelling time. Plots of the fraction of BrdUrd-labelled cells v. post-labelling time were then made for each window. T_S obtained in this way was in agreement with T_S obtained with the corrected RM method. In conclusion, we present a method to calculate T_s which theoretically first makes the determination of RM independent of T_G2+M, and secondly compensates for the non-linear function of RM with post-labelling time caused by accumulation of BrdUrd-labelled cells in G₂+ M.     

Bromodeoxyuridine: a diagnostic tool in biology and medicine, Part III. Proliferation in normal, injured and diseased tissue, growth factors, differentiation, DNA replication sites andin situ hybridization

Summary This paper is a continuation of parts I (history, methods and cell kinetics) and II (clinical applications and carcinogenesis) published previously (Dolbeare, 1995Histochem. J. 27, 339, 923). Incorporation of bromodeoxyuridine (BrdUrd) into DNA is used to measure proliferation in normal, diseased and injured tissue and to follow the effect of growth factors. Immunochemical detection of BrdUrd can be used to determine proliferative characteristics of differentiating tissues and to obtain birth dates for actual differentiation events. Studies are also described in which BrdUrd is used follow the order of DNA replication in specific chromasomes, DNA replication sites in the nucleus and to monitor DNA repair. BrdUrd incorporation has been used as a tool forin situ hybridization experiments.     

Cell-Cycle Analysis Using A Monoclonal Antibody to Brdurd

The flow cytometric measurement of DNA distributions of cells has many applications in biomedical research. Phase fractions estimated (calculated) from such distributions are used to study the growth characteristics of various types of cells, particularly when the cells have been exposed to perturbing agents such as chemotherapeutic drugs. For more than 10 years many methods for resolving DNA distributions into the three cell subpopulations (G₁, S and G₂, + M) have been reported in the literature. A new method of analysis utilizing a monoclonal antibody to bromodeoxyuridine (BrdUrd) has been developed (Gratzner, 1982; Dolbeare et al., 1983) which makes it possible in most cases to accurately determine phase fractions without resorting to mathematical models. the procedure involves the incorporation of BrdUrd by growing (DNA synthesizing) S phase cells, labelling the BrdUrd with a fluorescent monoclonal antibody, and the bivariate measurement of the antibody and of total DNA content, the latter through propidium-iodide staining. the resulting bivariate distributions clearly and simply resolve the three subpopulations. This paper describes the method and illustrates its use in the analysis of various fractions of elutriated exponentially growing Chinese hamster ovary (CHO) cells.     

Direct comparison of bromodeoxyuridine and Ki-67 labelling indices in human tumours

Direct comparison of bromodeoxyuridine (BrdUrd) and Ki-67 labelling indices was achieved by selecting similar areas from serial sections of human tumours. Fifteen patients were selected who had been administered BrdUrd in vivo and both proliferation markers were assessed by immunohistochemistry. The data show a good correlation between both BrdUrd LI and MIB-1 LI and T_pot (calculated using the flow cytometry derived duration of S phase) and MIB-1 LI. The contribution of BrdUrd LI to growth fraction varied as a function of proliferation characteristics. In tumours with a high LI, the number of DNA synthesizing cells represented half the growth fraction, whilst in tumours with lower LI's (<10%) the ratio of DNA precursor labelled cells as a function of growth fraction fell to between 10% and 20%. T_pot showed a linear correlation with MIB-1/BrdUrd ratio with a slope approaching unity. It was apparent that both intra- and interpatient variation in proliferation index was greater for BrdUrd labelling than for MIB-1 expression.     

The labelling index of human and mouse tumours assessed by bromodeoxyuridine staining in vitro and in vivo and flow cytometry

Bromodeoxyuridine (BrdUrd) incorporation and flow cytometry were used to measure human tumour kinetic parameters in vitro and in vivo. The technique was validated by comparison of labelling index estimates of mouse tumours in vivo and in vitro using BrdUrd and flow cytometry with tritiated thymidine (3HdThd) autoradiography. Similar labelling indices were obtained with both in vivo and in vitro incorporation into DNA of the two different precursors. Measurements of human tumour labelling indices were similar following in vitro incubation with either BrdUrd or 3HdThd. The use of BrdUrd allowed the visualization of a population of S-phase cells that did not appear to incorporate BrdUrd or 3HdThd. The human tumour labelling indices obtained with BrdUrd incorporation were similar to previously reported values using autoradiography studies. Preliminary studies demonstrated that significant human tumour labelling could be achieved with an intravenous injection of 500 mg BrdUrd.     

Induction of placental alkaline phosphatase in choriocarcinoma cells by 5-bromo-2′-deoxyuridine

Summary Growth of choriocarcinoma cells in the presence of 5-bromo-2′-deoxyuridine (BrdUrd) results in a 30- to 40-fold increase in alkaline phosphatase activity. The effect of BrdUrd is specific for phosphatase with an alkaline pH optimum. The induction by BrdUrd is probably not due to the production of an altered enzyme, since the induced enzyme resembles the basal enzyme in thermal denaturation and kinetic properties. Enzyme induction can be prevented by thymidine but not by deoxycytidine or deoxyuridine. The induction of alkaline phosphatase appears to require incorporation of the BrdUrd into cellular DNA. The presence of BrdUrd in the growth medium is not necessary for alkaline phosphatase induction in proliferating cells containing: BrdUrd-substituted genomes. However, enzyme induction and maintenance of the induced levels of alkaline phosphatase in nonproliferating cells containing BrdUrd-substituted DNA requires the presence of the analogues in the medium. The induction of alkaline phosphatase by BrdUrd in probably an indirect process.     

Flow cytometric measurement of cell cycle kinetics in rat Walker-256 carcinoma following in vivo and in vitro pulse labelling with bromodeoxyuridine.

Flow cytometric measurements of total DNA content, cell cycle distribution, and bromodeoxyuridine (BrdUrd) uptake were made in rat Walker-256 carcinoma cells. After both in vivo and in vitro pulse labelling with BrdUrd, Walker-256 tumor cells were stained with propidium iodide (PI) to estimate the total DNA content and a monoclonal antibody against BrdUrd to estimate the relative amount of cells in S phase. BrdUrd-labelled single cell suspensions were harvested at different time intervals to determine the movement of these cells within the cell cycle. To increase BrdUrd uptake, fluorodeoxyuridine (FDU), a thymidine antagonist, was also applied in vivo and in vitro. The results indicated exponential growth characteristics for this tumor between days 5 and 8 after implantation. Tumor doubling times, derived from changes in tumor volume in vivo and from the increase in cell number in vitro were similar. The mean time for DNA synthesis was estimated from the relative movement of BrdUrd-labelled cells towards G2. The percent of cells labelled with BrdUrd and the DNA synthesis time were similar regardless of the mode of BrdUrd administration. This study demonstrates that BrdUrd labelling of rat Walker-256 carcinoma cells in vitro yields kinetic estimates of tumor proliferation during exponential growth similar to those with the administration of BrdUrd in the intact tumor-bearing rat.     

Cell cycle analysis by the relative movement approach: effect of variability across S-phase of DNA synthesis rate

Cell populations pulse-labelled with BrdUrd, and sampled at increasing times after the pulse, yield DNA-BrdUrd distributions from which the relative movement (RM) and the depletion function (DF) of labelled, undivided cells can be calculated. In this paper we present an extension of the equation for the time course of RM, given by White and Meistrich (Cytometry 1986, 7 , 486–490), to the case in which the rate of DNA synthesis changes across S-phase. Some modalities of cell loss were also considered. Computer simulations showed that different patterns of DNA synthesis rate across S-phase can result in appreciably different RM curves. An analytical expression of the RM curve, in which the variability across S-phase of the rate of DNA synthesis is accounted for by only one parameter, was proposed. This expression was used for the simultaneous fitting of time sequences of RM and DF data of U937 cells, in order to estimate the phase transit times T_S and T_G2+M, and the potential doubling time T_pot. The use of the extended model gave better results than those obtained under the assumption of constant rate of DNA synthesis across S-phase.     

Induction of placental alkaline phosphatase in choriocarcinoma cells by 5-bromo-2'-deoxyuridine.

Growth of choriocarcinoma cells in the presence of 5-bromo-2'-deoxyuridine (BrdUrd) results in a 30- to 40-fold increase in alkaline phosphatase activity. The effects of BrdUrd is specific for phosphatase with an alkaline pH optimum. The induction by BrdUrd is probably not due to the production of an altered enzyme, since the induced enzyme resembles the basal enzyme in thermal denaturation and kinetic properties. Enzyme induction can be prevented by thymidine but not by deoxycytidine or deoxyuridine. The induction of alkaline phosphatase appears to require incorporation of the BrdUrd into cellular DNA. The presence of BrdUrd in the growth medium is not necessary for alkaline phosphatase induction in proliferating cells containing BrdUrd-substituted genomes. However, enzyme induction and maintenance of the induced levels of alkaline phosphatase in nonproliferating cells containing BrdUrd-substituted DNA requires the presence of the analogues in the medium. The induction of alkaline phosphatase by BrdUrd in probably an indirect process.     

Bromodeoxyuridine (BrdUrd) immunohistochemistry in undecalcified plastic-embedded tissue. Elimination of the DNA denaturation step

Summary We examined the application of BrdUrd immunohistochemistry to detect S-phase cells in undecalcified bone and cartilage from the growing rat embedded in Spurr's resin. The effect of fixation on the procedure was studied, and the validity of the technique examined by a comparative study with tritiated thymidine ([³H]-TdR) autoradiography. The use of sodium-ethoxide to remove plastic from tissue sections prior to immunohistochemistry resulted in the production of sufficient ssDNA to make a separate DNA denaturation step unnecessary, thus sparing sections from potentially destructive treatment and shortening the immunohistochemical procedure. Fixation in formalin or Bouin's fluid gave the most satisfactory results. The distribution of BrdUrd labeled cells was restricted to the sites of cell proliferation in growing long bones. Combined studies with BrdUrd immunohistochemistry and [³H]-TdR autoradiography showed that the majority of BrdUrd labeled cells had also incorporated [³H]-TdR, thus attesting to the validity of the technique. This novel approach is suitable for the study of undecalcified hard tissues as well as soft tissues.     

On the mechanism of 5-bromodeoxyuridine induction of prolactin synthesis in rat pituitary tumor cells

GH12C1, a clonal strain of rat pituitary tumor cells in culture (GH cells), does not produce detectable amounts of prolactin. 5-Bromodeoxyuridine (BrdUrd), the thymidine analogue, at sublethal concentrations (3-5 microgram/ml) induces prolactin synthesis in these cells. BrdUrd also induces prolactin synthesis in F1BGH12C1 cells, a BrdUrd resistant (BrdUrdr) substrain isolated from GH12C1 cells. The F1BGH12C1 strain is not drug dependent, but its resistance to BrdUrd is a stable phenotype. The significant features of the induction of prolactin synthesis in the BrdUrdr strain are the increased net synthesis of prolactin and the shortening of the lag period of prolactin induction. As BrdUrd concentration in the growth medium is increased, the rise in prolactin synthesis parallels the increased incorporation of BrdUrd into DNA. Prolactin synthesis is first detected when BrdUrd replaces 20-25% of the thymidine in DNA. BrdUrd can replace up to 75-80% of the thymidine within 2 d of treatment. Partial starvation of these cells under specified growth conditions does not affect the general growth pattern of the cells, general protein synthesis, and thymidine uptake, but does affect DNA synthesis. When cells are cultured under conditions in which DNA synthesis is preferentially inhibited, BrdUrd does not induce prolactin synthesis, suggestive of a DNA-mediated mechanism of action for the drug.     

Cell proliferation kinetics of six xenografted human cervix carcinomas: comparison of autoradiography and bromodeoxyuridine labelling methods

Abstract. Cell kinetic and histologic parameters of six xenografted tumours with volume doubling times ranging from 6 to 43 d were investigated in order to obtain kinetic information on a panel of tumours to be used in radiobiological studies. The six tumours covered a range of histologies and their DNA indices varied from 2–7 to 1–4. The length of the cell cycle (T_c), potential doubling time (T_pot) and labelling index (LI) were determined by continuous labelling with [³H]TdR and autoradiography in three tumours. T_c varied from 30 to 40 h. Determinations of the length of the S phase (T_s) were found to be less reliable by this method. Data on T_s and LI were also determined in all six tumours using bromodeoxyuridine (BrdU) labelling and the single sample method; values of T_pot were slightly longer than those obtained via the autoradiographic method. In addition, multiple samples were taken after BrdU labelling. T_c was determined by fitting the data obtained from mid-S, mid-G₂ and mid-G₁ windows to curves described by a damped oscillator. Data obtained via the mid-S window were found to be most reliable. Generally, cell cycle times obtained by the BrdU method were longer than those observed with the autoradiographic method. Differences between the two methods could be explained by inaccuracies in the determination of T_s, LI and T_c and differences in the experimental approach. We consider the BrdU labelling method to be a suitable alternative for the time-consuming autoradiography, if data on T_s or T_pot are sufficient. Due to difficulties in the reproducibility of the immunofluorescence staining and asynchronization of cells approximately 10 h after labelling, the method of windows analysis was affected by similar problems to those observed in interpretation of percentage labelled mitosis (PLM) curves. However, the method may serve as an alternative to determine cell cycle times in vitro and, if improved technically, in vivo. Careful comparison of the data obtained from mid-S, mid-G₁ and mid-G₂ windows may increase the reliability of the determination of cell kinetic parameters.     

A method to measure the duration of DNA synthesis and the potential doubling time from a single sample

A method is described whereby the DNA synthesis time, Ts, can be calculated using data of a single sample of cells taken several hours after labelling with bromodeoxyuridine (BrdUrd). The method involves a simple calculation using flow cytometry data of BrdUrd incorporation (green fluorescence, FITC-labelled anti-BrdUrd-DNA antibody) and total DNA content (red fluorescence, propidium iodide). The movement of BrdUrd-labelled cells through the S phase can be quantified by measuring their mean red fluorescence relative to that of G1 and G2 cells. Assuming the movement of the labelled cells toward G2 is linear with time, Ts can be calculated by measuring their relative movement at any one time. The method was tested on cells in vitro and on bone marrow and tumor cells in vivo. Reasonable agreement was seen with published estimates of Ts for these tissues.     

Validation of bromodeoxyuridine immunohistochemistry for localization of S-phase cells in decalcified tissues. A comparative study with tritiated thymidine autoradiography

Summary Immunohistochemical detection of the thymidine analogue 5-bromo-2-deoxyuridine (BrdUrd), which is incorporated by S-phase cells, offers a convenient way of studying the proliferation kinetics of cells in normal skeletal tissues and in bone containing/derived tumours. To assess the validity of using this approach on decalcified, paraffin embedded tissues, the BrdUrd method was compared with tritiated thymidine (³H-TdR) autoradiography, using rat tibiae labelled with both³H-TdR and BrdUrd, fixed in Carnoy's fluid and decalcified in EDTA, prior to routine paraffin embedding. The distribution of BrdUrd-labelled cells correlated with the sites of cell proliferation in the growing rat tibia.Independent studies with each method on paired serial sections of double-labelled tissue, showed a highly significant correlation (r=0.81, p<0.0003) in the numbers of labelled cells seen in autoradiographs and immunostained sections from the proximal tibial growth plate. Combined BrdUrd immunohistochemistry and³H-TdR autoradiography showed that the majority of labelled cells in cartilage, bone marrow, and fibrous perichondrium and periosteum had incorporated both labels. These results show that BrdUrd immunohistochemistry is a valid technique for the study of dividing cells in mineralized tissues after decalcification.     

Flow cytometric estimation of cell cycle parameters using a monoclonal antibody to bromodeoxyuridine

An estimation of cell kinetic parameters was made by simultaneous flow cytometric measurements of DNA and bromodeoxyuridine (BrdUrd) contents of cells. The procedure described in this paper involves the incorporation of BrdUrd by S phase cells, labeling the BrdUrd with an indirect immunofluorescent technique using a monoclonal anti-BrdUrd antibody, and staining DNA with propidium iodide (PI). The amount of incorporated BrdUrd in HeLa cells was proportional to that of synthesized DNA through S phase. For all cell lines examined, the pattern of BrdUrd incorporation was essentially the same and the rate of DNA synthesis during S phase was not constant. The bivariate BrdUrd/DNA distributions showed a horse-shoe pattern, maximum in the mid S phase and minimum in the early and late S phases. Furthermore, the durations of cell cycle (Tc) and S phase (Ts) were estimated from a FLSm (fraction of labeled cells in mid S phase) curve that was generated by plotting the percentage of BrdUrd pulse-labeled cells in a narrow window defined in the mid S phase of the DNA histogram. The values of these parameters in NIH 3T3, HeLa S3, and HL-60 cells were in good accordance with the reported data. This FCM method using the monoclonal anti-BrdUrd antibody allows rapid determination of both cell cycle compartments and also Ts and Tc without the use of radioactive DNA precursors.     

Mutational and pseudomutational effects of 5-bromodeoxyuridine in human lymphoblasts

We have studied the effects of 5-bromodeoxyuridine (BrdUrd) at two genetic loci in diploid human lymphoblast cells. In thymidine kinase heterozygotes (tk +/-), a 2-h dose of BrdUrd induced a transient, non-heritable resistance to the thymidine analogue, trifluorothymidine (F3TdR). We have called this phenomenon pseudomutation and have shown that affected cells acquire the ability to survive in the presence of F3TdR and then, after degradation of F3TdR in the medium, return to an apparently normal wild-type state. Our data suggest that BrdUrd incorporation into DNA as a thymidine analogue is responsible for the effect, which we interpret as a temporary loss of thymidine kinase activity. This effect is not seen in tk +/+ homozygotes. In contrast, at the hypoxanthine-guanine phosphoribosyl transferase locus in tk +/- heterozygotes, BrdUrd did not induce a permanent, heritable resistance to 6-thioguanine (gene locus mutation). We detected such mutations only in the tk +/+ homozygote and only at external BrdUrd concentrations considerably higher than those which saturate the uptake of BrdUrd into DNA as a thymidine analogue. We postulate that the reduced TK enzyme levels (30%) in the heterozygote prevent the build-up of a sufficiently high intracellular BrdUrd triphosphate pool to promote the misincorporations as deoxycytidine triphosphate which may be responsible for gene locus mutation.     

Characterization of monoclonal antibodies to bromodeoxyuridine

The characteristics of three mouse monoclonal antibodies to halogenated uridine derivatives are presented. Two, IU-1 and IU-2, are produced by hybridomas derived in our laboratory, and the third is the B-44 hybridoma described by Gratzner (7) and obtained commercially from Becton-Dickinson Monoclonal Center. Hybridomas IU-1 and IU-2 were derived from the fusion of spleen cells from a Biozzi High Responder mouse immunized with iododeoxyuridine (IdUrd) conjugated to bovine serum albumin and SP2/0 mouse myeloma cells. This paper presents methods and results for enzyme-linked immunosorbent assays (ELISA) against whole cells labeled with bromodeoxyuridine (BrdUrd), ELISA against BrdUrd-labeled DNA, and a competition ELISA for free BrdUrd. All three antibodies show similar binding affinities and specificities. The IU antibodies react with BrdUrd and IdUrd when the nucleosides are either free in solution or incorporated into single-stranded DNA (ss-DNA). The antibodies do not recognize either halogenated base in double-stranded DNA (ds-DNA), nor do they react with uracil or bromocytidine. Weak binding to thymidine, 5-fluorodeoxyuridine, and unsubstituted ss-DNA occurs.     

Molecular interaction studies of zinc sulphide nanoparticles with DNA and its consequence: a multitechnique approach

Molecular interaction studies between nanoparticles (NPs) and biomolecules are of great importance in the field of nanomedicine as they affect many physiological processes. Therefore, the interaction of zinc sulphide nanoparticles (ZnS NPs) with calf thymus deoxyribonucleic acid (CT DNA) and its significance was analyzed using ultraviolet (UV)–visible light, fluorescence, circular dichroism (CD), zeta potential, viscometry, electrochemical, and polymerase chain reaction methods. Fluorescence quenching analysis revealed that the fluorescence of ZnS NPs was quenched using CT DNA through a static quenching mechanism. The negative values of thermodynamic parameters (ΔG, ΔH, and ΔS) showed that the binding process was spontaneous, exothermic, and van der Waals or hydrogen bonding plays an important role in the interaction of ZnS NPs with CT DNA. Thermal melting (T_m) studies indicated a decrease in the T_m of CT DNA, suggesting the destabilization of CT DNA upon interaction with ZnS NPs. In addition, the results obtained from competitive binding, zeta potential, CD, and viscometry measurements showed that the interaction of ZnS NPs with CT DNA is through groove binding. Electrochemical analysis further confirmed the observed results from various spectroscopic and other related studies, in which decrease in the redox peak current along with changes in peak potential (CV) and increase in the electrical resistance (EIS) indicated the interaction between ZnS NPs and CT DNA. Furthermore, PCR analysis using DNA polymerase revealed the potential of ZnS NPs to inhibit DNA replication in vitro. ZnS NP–CT DNA interaction studies can be explored to define new horizons in biomedical applications of ZnS NPs.     

Comparison of isotopic and immunohistochemical methods of studying epithelial cell proliferation in hamster tongue

Our purpose was to validate different approaches to the study of cell proliferation in stratified squamous epithelia, using oral mucosa as a model. Dorsal and ventral tongue from the hamster were examined following in vivo labelling with tritiated thymidine and bromodeoxyuridine (BrdUrd), and in vitro labelling with BrdUrd. These were compared with direct immunolabelling of fixed tissue sections with monoclonal antibody PC10. For the former methods S phase cells were quantified following autoradiography or immunohistochemistry. We conclude that the proliferative status of simple, flat, lining mucosae such as ventral tongue can be derived by all three prelabelling methods and, on average, 18–19 cells per surface millimetre length were in DNA synthesis. On the other hand dorsal tongue epithelium, which is thicker, has an undulating morphology and a complex cell renewal pattern, gives different results with the three labelling methods. In both sites the proliferating cell nuclear antigen (PCNA) index was fourfold that obtained by nucleotide labelling. This is consistent with PCNA marking proliferative cells in other phases of the cell cycle in addition to the S phase. Thus, there are potential differences between the information on proliferative status derived by PCNA immunohistochemistry and other established cell cycle markers, which need to be taken into account in the interpretation of epithelial cell kinetic data in health and disease.     

Reductive transformation of TNT by Escherichia coli resting cells: kinetic analysis

Microbial 2,4,6-trinitrotoluene (TNT) biotransformation via sequential nitro-reduction appears a ubiquitous process, but the kinetics of these transformations have been poorly understood or described. TNT transformation by Escherichia coli was monitored and a kinetic model for reductive TNT depletion was developed and experimentally calibrated in this report. Using resting cells of aerobically pregrown E. coli, TNT was quickly reduced to hydroxylaminodinitrotoluenes. The standard Michaelis–Menten model was modified to include three additional parameters: product toxicity (T _c), substrate inhibition (K _i), and intracellular reducing power (RH) limitation. Experimentally measured product toxicity (5.2 μmol TNT/mg cellular protein) closely matched the best-fit model value (2.84 μmol TNT/mg cellular protein). Parameter identifiability and reliability (k _m, K _s, T _c, and K _i) was evaluated and confirmed through sensitivity analyses and via Monte Carlo simulations. The resulting kinetic model adequately described TNT reduction kinetics by E. coli resting cells in the absence or presence of reducing power limitation.