Kinetics of in vivo proliferation and death of memory and naive CD8 T cells: parameter estimation based on 5-bromo-2'-deoxyuridine incorporation in spleen, lymph nodes, and bone marrow

To study naive and memory CD8 T cell turnover, we performed BrdU incorporation experiments in adult thymectomized C57BL/6 mice and analyzed data in a mathematical framework. The following aspects were novel: 1) we examined the bone marrow, in addition to spleen and lymph nodes, and took into account the sum of cells contained in the three organs; 2) to describe both BrdU-labeling and -delabeling phase, we designed a general mathematical model, in which cell populations were distinguished based on the number of divisions; 3) to find parameters, we used the experimentally determined numbers of total and BrdU(+) cells and the BrdU-labeling coefficient. We treated mice with BrdU continuously via drinking water for up to 42 days, measured by flow cytometry BrdU incorporation at different times, and calculated the numbers of BrdU(+) naive (CD44(int/low)) and memory (CD44(high)) CD8 T cells. By fitting the model to data, we determined proliferation and death rates of both subsets. Rates were confirmed using independent sets of data, including the numbers of BrdU(+) cells at different times after BrdU withdrawal. We found that both doubling time and half-life of the memory population were approximately 9 wk, whereas for the naive subset the doubling time was almost 1 year and the half-life was roughly 7 wk. Our findings suggest that the higher turnover of memory CD8 T cells as compared with naive CD8 T cells is mostly attributable to a higher proliferation rate. Our results have implications for interpreting physiological and abnormal T cell kinetics in humans.     

Estimating average cellular turnover from 5-bromo-2'-deoxyuridine (BrdU) measurements

Cellular turnover rates in the immune system can be determined by labelling dividing cells with 5-bromo-2'-deoxyuridine (BrdU) or deuterated glucose ((2)H-glucose). To estimate the turnover rate from such measurements one has to fit a particular mathematical model to the data. The biological assumptions underlying various models developed for this purpose are controversial. Here, we fit a series of different models to BrdU data on CD4(+) T cells from SIV(-) and SIV(+) rhesus macaques. We first show that the parameter estimates obtained using these models depend strongly on the details of the model. To resolve this lack of generality we introduce a new parameter for each model, the 'average turnover rate', defined as the cellular death rate averaged over all subpopulations in the model. We show that very different models yield similar estimates of the average turnover rate, i.e. ca. 1% day(-1) in uninfected monkeys and ca. 2% day(-1) in SIV-infected monkeys. Thus, we show that one can use BrdU data from a possibly heterogeneous population of cells to estimate the average turnover rate of that population in a robust manner.     

Modeling reveals that dynamic regulation of c-FLIP levels determines cell-to-cell distribution of CD95-mediated apoptosis

The expression levels of caspase-8 inhibitory c-FLIP proteins play an important role in regulating death receptor-mediated apoptosis, as their concentration at the moment when the death-inducing signaling complex (DISC) is formed determines the outcome of the DISC signal. Experimental studies have shown that c-FLIP proteins are subject to dynamic turnover and that their stability and expression levels can be rapidly altered. Even though the influence of c-FLIP on the apoptotic behavior of a single cell has been captured in mathematical simulation studies, the effect of c-FLIP turnover and stability has not been investigated. In this study, a mathematical model of apoptosis was developed to analyze how the dynamic turnover and stability of the c-FLIP isoforms regulate apoptotic signaling for both individual cells and cell populations. Intercellular parameter and concentration distributions were used to describe the behavior of cell populations. Monte-Carlo simulations of cell populations showed that c-FLIP turnover is a key determinant of death receptor responses. The fact that the developed model simulates the state of whole cell populations makes it possible to validate it by comparison with empirical data. The proposed modeling approach can be used to further determine limiting factors in the DISC signaling process.     

Cell turnover in the vomeronasal epithelium: evidence for differential migration and maturation of subclasses of vomeronasal neurons in the adult opossum

Previous investigations of cell turnover in the mammalian vomeronasal sensory epithelium (VN-SE) raised two issues. First, if, in addition to the already demonstrated vertical migration, horizontal migration from the edges of the VN-SE participates in neuronal replacement. Second, whether or not migration and maturation is differential in upper and lower populations of vomeronasal neurons, since these two cell populations are chemically, physiologically, functionally, and perhaps evolutionarily different. By injecting bromodeoxyuridine (BrdU) into adult opossum (Monodelphis domestica) and permitting different survival times, the pattern of distribution of BrdU-labeled cells was analyzed. No evidence of horizontal migration in neuronal replacement was found. To investigate vertical migration and maturation of subclasses of vomeronasal neurons, double immunohistochemistry of BrdU and markers of the lower (G(oalpha) protein) and upper [G(i2alpha) protein and olfactory marker protein (OMP)] cell populations were performed. Three days after administration of BrdU, some mature neurons were observed in both lower and upper layers of the VN-SE, as demonstrated by coexpression of BrdU with G(oalpha) protein and OMP, respectively. The data on vertical distribution, however, indicate that most of the daughter cells enter the G(oalpha)-protein-expressing zone of the VN-SE by day 5, whereas most daughter cells do not reach the G(i2alpha)-protein-expressing zone until day 7, suggesting that these two populations mature at slightly different rates. These results are the first evidence of differential neurogenesis of subclasses of vomeronasal neurons.     

Mitogen induction of murine C-type viruses. I. Analysis of lymphoid cell subpopulations.

We reported previously in vitro induction of endogenous C-type viruses from normal mouse spleen cells by lipopolysaccharide (LPS) as well as by combination treatment with concanabalin A and 5-bromo-2'-deoxyuridine (Con A/BrdU). To identify the cell types sensitive to virus induction and to study the relationship of mitogenicity to virus induction we have compared T cell populations (BALB/c thymus cells and cortisone-resistant thymus cells), B cell populations (nu/nu spleen cells and lymph node cells), adherent BALB/c peritoneal cells and mixed populations (BALB/c spleen cells, macrophage-depleted BALB/c spleen cells, and lymph node cells). LPS-induction occurred only in B cell-containing populations. In contrast, induction by Con A/BrdU depended on the presence of both T and B cells. In both instances, neither macrophages nor hemopoietic cells appeared to be a major source of virus. Treatment with anti-Ig serum and complement reduced virus induction by LPS/BrdU but not by Con A/BrdU suggesting that different cell populations produce virus after stimulation with these two different mitogens.     

PCNA/cyclin expression and BrdU uptake define different subpopulations in different cell lines.

Monoclonal antibodies (MAb) to a 36 KD protein, proliferating cell nuclear antigen (PCNA/cyclin), have been previously shown to be capable of identifying proliferating cells in vitro as well as in alcohol-fixed, paraffin-embedded tissue specimens. The routine use of these anti-PCNA/cyclin MAb in investigative studies and in diagnostic pathology requires a clearer understanding of the distribution of PCNA/cyclin in the different cell populations found in tissue specimens. We therefore compared the ability of MAb to three nucleus-associated proliferation markers (MAb 19A2 to PCNA/cyclin; Ki-67 to an undefined proliferation-related marker; BU-1 to 5'-bromodeoxyuridine (BrdU) incorporated into DNA) to identify the proliferating cell fraction of various cells in vitro. The cell lines were chosen to represent a spectrum of proliferation rates (high to low) and cell lineage (mesenchymal vs epithelial, non-transformed vs malignant): (a) HeLa and A-431 (two malignant carcinoma cell lines with high proliferation rates); (b) SK-5 (a non-transformed fibroblast cell line with a low proliferation rate); (c) HUVE (a non-transformed human umbilical vein endothelial cell line with a low proliferation rate). Single and double labeling immunofluorescence studies were performed after uniform 1-hr incubations with BrdU. Comparison of the overlapping distributions of detectable PCNA/cyclin expression and BrdU incorporation demonstrated substantial qualitative and quantitative differences between the different cell lines. In two of the four cell lines (HeLa, A-431) the BrdU staining distributions formed inclusive subsets of the PCNA-positive cell populations. In the HUVE cell line the two populations overlapped incompletely. In one cell line, SK-5, the two populations were mutually exclusive. MAb Ki-67 demonstrated a pattern in the SK-5 cell line that was strongly predictive of PCNA positivity, while showing no associated patterns in the other three cell lines. We conclude that PCNA/cyclin expression detected by MAb may define different cell subpopulations in different cell types relative to those incorporating BrdU or expressing the target antigen for Ki-67. This has implications for the clinical study of mixed cell populations using these antibodies.     

Estimating Dormant and Active Hematopoietic Stem Cell Kinetics through Extensive Modeling of Bromodeoxyuridine Label-Retaining Cell Dynamics

Bone marrow hematopoietic stem cells (HSCs) are responsible for both lifelong daily maintenance of all blood cells and for repair after cell loss. Until recently the cellular mechanisms by which HSCs accomplish these two very different tasks remained an open question. Biological evidence has now been found for the existence of two related mouse HSC populations. First, a dormant HSC (d-HSC) population which harbors the highest self-renewal potential of all blood cells but is only induced into active self-renewal in response to hematopoietic stress. And second, an active HSC (a-HSC) subset that by and large produces the progenitors and mature cells required for maintenance of day-to-day hematopoiesis. Here we present computational analyses further supporting the d-HSC concept through extensive modeling of experimental DNA label-retaining cell (LRC) data. Our conclusion that the presence of a slowly dividing subpopulation of HSCs is the most likely explanation (amongst the various possible causes including stochastic cellular variation) of the observed long term Bromodeoxyuridine (BrdU) retention, is confirmed by the deterministic and stochastic models presented here. Moreover, modeling both HSC BrdU uptake and dilution in three stages and careful treatment of the BrdU detection sensitivity permitted improved estimates of HSC turnover rates. This analysis predicts that d-HSCs cycle about once every 149–193 days and a-HSCs about once every 28–36 days. We further predict that, using LRC assays, a 75%–92.5% purification of d-HSCs can be achieved after 59–130 days of chase. Interestingly, the d-HSC proportion is now estimated to be around 30–45% of total HSCs - more than twice that of our previous estimate.     

Evaluation of intranuclear BrdU detection procedures for use in multicolor flow cytometry.

BACKGROUND: Measurement of cell proliferation via BrdU incorporation in combination with multicolor cell surface staining would facilitate studies on cell subsets that require multiple markers for their identification. However, the extent to which the often harsh cell preparation procedures required affect the staining quality of more recently developed fluorescent dyes has not been assessed. METHODS: Three cell preparation protocols for BrdU measurement were compared for their ability to maintain fluorescent surface staining and scatter parameters of in vivo BrdU-labeled cells by flow cytometry. A 10-color fluorescent panel was developed to test the quality of surface staining, following cell treatment and the ability to perform BrdU measurements on even small B lymphocyte subsets. RESULTS: All cell preparation procedures affected the quality of fluorescent and/or scatter parameters to varying degrees. Paraformaldehyde/saponin-based procedures preserved sufficient fluorescent surface staining to determine BrdU incorporation rates among all splenic B cell subsets, including B-1a cells, which constitute roughly 0.5% of cells. Turnover rates of B-1a cells were similar to immature B cells and higher than those of the other mature B cell subsets. CONCLUSION: Paraformaldehyde/saponin-based cell preparation procedures facilitate detailed cell turnover studies on small cell subsets in vivo, revealing new functional information on rare cell populations.     

Reduced cell turnover in bovine leukemia virus-infected, persistently lymphocytotic cattle

Although nucleotide analogs like bromodeoxyuridine have been extensively used to estimate cell proliferation in vivo, precise dynamic parameters are scarce essentially because of the lack of adequate mathematical models. Besides recent developments on T cell dynamics, the turnover rates of B lymphocytes are largely unknown particularly in the context of a virally induced pathological disorder. Here, we aim to resolve this issue by determining the rates of cell proliferation and death during the chronic stage of the bovine leukemia virus (BLV) infection, called bovine persistent lymphocytosis (PL). Our methodology is based on direct intravenous injection of bromodeoxyuridine in association with subsequent flow cytometry. By this in vivo approach, we show that the death rate of PL B lymphocytes is significantly reduced (average death rate, 0.057 day(-1) versus 0.156 day(-1) in the asymptomatic controls). Concomitantly, proliferation of the PL cells is also significantly restricted compared to the controls (average proliferation rate, 0.0046 day(-1) versus 0.0085 day(-1)). We conclude that bovine PL is characterized by a decreased cell turnover resulting both from a reduction of cell death and an overall impairment of proliferation. The cell dynamic parameters differ from those measured in sheep, an experimental model for BLV infection. Finally, cells expressing p24 major capsid protein ex vivo were not BrdU positive, suggesting an immune selection against proliferating virus-positive lymphocytes. Based on a comparative leukemia approach, these observations might help to understand cell dynamics during other lymphoproliferative disease such as chronic lymphocytic leukemia or human T-cell lymphotropic virus-induced adult T-cell leukemia in humans.     

Bromodeoxyuridine specifically labels the regenerative stem cells of planarians

The singular regenerative abilities of planarians require a population of stem cells known as neoblasts. In response to wounding, or during the course of cell turnover, neoblasts are signaled to divide and/or differentiate, thereby replacing lost cell types. The study of these pluripotent stem cells and their role in planarian regeneration has been severely hampered by the reported inability of planarians to incorporate exogenous DNA precursors; thus, very little is known about the mechanisms that control proliferation and differentiation of this stem cell population within the planarian. Here we show that planarians are, in fact, capable of incorporating the thymidine analogue bromodeoxyuridine (BrdU), allowing neoblasts to be labeled specifically during the S phase of the cell cycle. We have used BrdU labeling to study the distribution of neoblasts in the intact animal, as well as to directly demonstrate the migration and differentiation of neoblasts. We have examined the proposal that a subset of neoblasts is arrested in the G2 phase of the cell cycle by double-labeling with BrdU and a mitosis-specific marker; we find that the median length of G2 (approximately 6 h) is sufficient to account for the initial mitotic burst observed after feeding or amputation. Continuous BrdU-labeling experiments also suggest that there is not a large, slow-cycling population of neoblasts in the intact animal. The ability to label specifically the regenerative stem cells, combined with the recently described use of double-stranded RNA to inhibit gene expression in the planarian, should serve to reignite interest in the flatworm as an experimental model for studying the problems of metazoan regeneration and the control of stem cell proliferation.     

Synchronized oscillations in growing cell populations are explained by demographic noise

Understanding synchrony in growing populations is important for applications as diverse as epidemiology and cancer treatment. Recent experiments employing fluorescent reporters in melanoma cell lines have uncovered growing subpopulations exhibiting sustained oscillations, with nearby cells appearing to synchronize their cycles. In this study, we demonstrate that the behavior observed is consistent with long-lasting transient phenomenon initiated and amplified by the finite-sample effects and demographic noise. We present a novel mathematical analysis of a multistage model of cell growth, which accurately reproduces the synchronized oscillations. As part of the analysis, we elucidate the transient and asymptotic phases of the dynamics and derive an analytical formula to quantify the effect of demographic noise in the appearance of the oscillations. The implications of these findings are broad, such as providing insight into experimental protocols that are used to study the growth of asynchronous populations and, in particular, those investigations relating to anticancer drug discovery.     

Stem Cell Proliferation and Quiescence—Two Sides of the Same Coin

The kinetics of label uptake and dilution in dividing stem cells, e.g., using Bromodeoxyuridine (BrdU) as a labeling substance, are a common way to assess the cellular turnover of all hematopoietic stem cells (HSCs) in vivo. The assumption that HSCs form a homogeneous population of cells which regularly undergo cell division has recently been challenged by new experimental results. For a consistent functional explanation of heterogeneity among HSCs, we propose a concept in which stem cells flexibly and reversibly adapt their cycling state according to systemic needs. Applying a mathematical model analysis, we demonstrate that different experimentally observed label dilution kinetics are consistently explained by the proposed model. The dynamically stabilized equilibrium between quiescent and activated cells leads to a biphasic label dilution kinetic in which an initial and pronounced decline of label retaining cells is attributed to faster turnover of activated cells, whereas a secondary, decelerated decline results from the slow turnover of quiescent cells. These results, which support our previous model prediction of a reversible activation/deactivation of HSCs, are also consistent with recent findings that use GFP-conjugated histones as a label instead of BrdU. Based on our findings we interpret HSC organization as an adaptive and regulated process in which the slow activation of quiescent cells and their possible return into quiescence after division are sufficient to explain the simultaneous occurrence of self-renewal and differentiation. Furthermore, we suggest an experimental strategy which is suited to demonstrate that the repopulation ability among the population of label retaining cells changes during the course of dilution.     

Stem Cell Proliferation and Quiescence—Two Sides of the Same Coin

The kinetics of label uptake and dilution in dividing stem cells, e.g., using Bromodeoxyuridine (BrdU) as a labeling substance, are a common way to assess the cellular turnover of all hematopoietic stem cells (HSCs) in vivo. The assumption that HSCs form a homogeneous population of cells which regularly undergo cell division has recently been challenged by new experimental results. For a consistent functional explanation of heterogeneity among HSCs, we propose a concept in which stem cells flexibly and reversibly adapt their cycling state according to systemic needs. Applying a mathematical model analysis, we demonstrate that different experimentally observed label dilution kinetics are consistently explained by the proposed model. The dynamically stabilized equilibrium between quiescent and activated cells leads to a biphasic label dilution kinetic in which an initial and pronounced decline of label retaining cells is attributed to faster turnover of activated cells, whereas a secondary, decelerated decline results from the slow turnover of quiescent cells. These results, which support our previous model prediction of a reversible activation/deactivation of HSCs, are also consistent with recent findings that use GFP-conjugated histones as a label instead of BrdU. Based on our findings we interpret HSC organization as an adaptive and regulated process in which the slow activation of quiescent cells and their possible return into quiescence after division are sufficient to explain the simultaneous occurrence of self-renewal and differentiation. Furthermore, we suggest an experimental strategy which is suited to demonstrate that the repopulation ability among the population of label retaining cells changes during the course of dilution.     

Determination of the fraction of S-phase cells in root meristems using bromodeoxyuridine labeling

The use of bromodeoxyuridine (BrdU) and subsequent immunocytochemical visualization for studying cell proliferation in plant meristems was investigated in Allium cepa L. root-tips. We describe the optimization of an indirect immunoperoxidase method for detecting incorporation of this DNA precursor in pulse-labeled cells. The basic object of this study is to quantify the extent to which the fraction of S-phase cells can reliably be estimated in asynchronous populations. A matrix of parallel labeling schedules with tritiated-thymidine or BrdU was developed, and the labeling indices provided by autoradiography or immunocytochemistry were compared. Thus, 0.5 mM BrdU assured saturation S-phase labeling after an exposure time of 30 min, and the mean length of the S-phase determined under such conditions was similar to that previously reported for this plant system. Interestingly, Feulgen staining did not interfere with subsequent detection of the BrdU probe. This allowed comparative evaluations of the nuclear DNA content by Feulgenmicrodensitometry and the position of a given cell in G1, S or G2 compartments. We also explored the possibility of quantifying BrdU-incorporation in single nuclei by densitometry measurement of the peroxidase label.     

Determination of the fraction of S-phase cells in root meristems using bromodeoxyuridine labeling

The use of bromodeoxyuridine (BrdU) and subsequent immunocytochemical visualization for studying cell proliferation in plant meristems was investigated in Allium cepa L. root-tips. We describe the optimization of an indirect immunoperoxidase method for detecting incorporation of this DNA precursor in pulse-labeled cells. The basic object of this study is to quantify the extent to which the fraction of S-phase cells can reliably be estimated in asynchronous populations. A matrix of parallel labeling schedules with tritiated-thymidine or BrdU was developed, and the labeling indices provided by autoradiography or immunocytochemistry were compared. Thus, 0.5 mM BrdU assured saturation S-phase labeling after an exposure time of 30 min, and the mean length of the S-phase determined under such conditions was similar to that previously reported for this plant system. Interestingly, Feulgen staining did not interfere with subsequent detection of the BrdU probe. This allowed comparative evaluations of the nuclear DNA content by Feulgenmicrodensitometry and the position of a given cell in G1, S or G2 compartments. We also explored the possibility of quantifying BrdU-incorporation in single nuclei by densitometry measurement of the peroxidase label.     

Hoechst fluorescence intensity can be used to separate viable bromodeoxyuridine-labeled cells from viable non-bromodeoxyuridine-labeled cells

BACKGROUND: 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells. METHODS: Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry. RESULTS: Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. CONCLUSIONS: Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability.     

The diverse effects of 5'-bromodeoxyuridine on enzyme activities in cultured H35 hepatoma cells.

Reuber (H35) hepatoma cells were grown in medium containing 10(-5)M bromodeoxyuridine (BrdU), which was incorporated into their DNA. Cell growth rate was unaffected by BrdU for the first two generations, after which it was reduced by about 50%. The effect of BrdU incorporation on the activities of several enzymes with rapid turnover rates was examined to test the hypothesis that the synthesis of such enzymes will be preferentially inhibited by BrdU. Tyrosine amino-transferase (TAT) activity decreased by 70% within two generations whereas thymidine kinase activity remained at control values. PEP carboxykinase activity was unchanged during the first generation in BrdU-containing medium but, during the second, its activity increased by at least 30%. Ornithine decarboxylase levels decreased by about 50% only after two generations in the presence of BrdU. There appeared to be no simple relationship between turnover rates and the effect of BrdU on enzyme activity. Incorporation of BrdU was found to inhibit the induction of both TAT and PEP carboxykinase by dexamethasone and to enhance the inhibition of cell growth by this steroid. These results are discussed with respect to possible mechanisms of gene expression and development in both normal and neoplastic cells.     

Analytical errors in measuring radioactivity in cell proteins and their effect on estimates of protein turnover in L cells.

Previous studies from this laboratory on protein turnover in 3H-labelled L-cell cultures have shown recovery of total 3H at the end of a 3-day experiment to be always significantly in excess of the 3H recovered at the beginning of the experiment. In this study we have critically reviewed a number of possible sources for this error in measuring radioactivity in cell proteins. 3H-labelled proteins, when dissolved in 0.3 M-NaOH and counted for radioactivity in a liquid-scintillation spectrometer, showed losses of 30-40% of the radioactivity; neither external or internal standardization compensated for this loss. Hydrolysis of these proteins with either Pronase or concentrated HCl significantly increased the measured radioactivity. In addition, approx. 5-10% of the cell protein is left on the plastic culture dish when cells are recovered in phosphate-buffered saline. To aggravate this latter loss further, this surface-adherent protein, after pulse labelling, contains proteins of high radioactivity that turn over rapidly and make a major contribution to the accumulating radioactivity in the medium. These combined errors can account for up to 60% of the total radioactivity in the cell culture. Similar analytical errors have been found in studies of other cell cultures. The effect of these analytical errors on estimates of protein turnover in cell cultures is discussed.     

Mathematical Determination of Cell Population Doubling Times for Multiple Cell Lines

Cell cycle times are vital parameters in cancer research, and short cell cycle times are often related to poor survival of cancer patients. A method for experimental estimation of cell cycle times, or doubling times of cultured cancer cell populations, based on addition of paclitaxel (an inhibitor of cell division) has been proposed in literature. We use a mathematical model to investigate relationships between essential parameters of the cell division cycle following inhibition of cell division. The reduction in the number of cells engaged in DNA replication reaches a plateau as the concentration of paclitaxel is increased; this can be determined experimentally. From our model we have derived a plateau log reduction formula for proliferating cells and established that there are linear relationships between the plateau log reduction values and the reciprocal of doubling times (i.e. growth rates of the populations). We have therefore provided theoretical justification of an important experimental technique to determine cell doubling times. Furthermore, we have applied Monte Carlo experiments to justify the suggested linear relationships used to estimate doubling time from 5-day cell culture assays. We show that our results are applicable to cancer cell populations with cell loss present.     

The effects of prolonged administration of 5-bromodeoxyuridine on cells of the immune system

We have determined the in vivo effect of 5-bromodeoxyuridine (BrdU) administered to mice in the drinking water for various lengths of time on the performance of T and B lymphocytes in a number of experimental protocols. Young mice continuously exposed to BrdU fail to gain weight, and the lymphocytes recovered after a prolonged period of exposure are fewer in number than in control mice. The recovery of normal levels of T and B lymphocytes after irradiation is severely impaired. Ag-specific cells responding to Ag in an adoptive transfer model fail to expand as much in the presence of BrdU as in the absence, and the Ag-specific effectors produced in the presence of BrdU are less able to secrete cytokines upon restimulation in vitro. Polarized populations of Tc1 and Tc2 effectors generated in vitro proliferate less in the presence of BrdU, and the resulting effectors make less cytokines per cell upon restimulation. Thus, the incorporation of BrdU into T or B lymphocytes can, under some circumstances, seriously impair the performance of the labeled cells, and these findings raise a note of caution in the interpretation of studies that make use of long-term exposure to BrdU.