DNA-synthesizing cells in oral epithelium have a range of cell cycle durations: evidence from double-labelling studies using tritiated thymidine and bromodeoxyuridine

Mouse tongue epithelium is characterized by a circadian variation in the number of DNA-synthesizing cells (labelling index, LI). Cells undergoing DNA synthesis were labelled with tritiated thymidine [( 3H]TdR) at 0300 (peak LI) or 1200 h (low LI). The fate of these cells was assessed by injecting animals with bromodeoxyuridine (BrdU) at intervals from 12-48 h after [3H]TdR, to follow them from one cell cycle to the next. Labelling was revealed by combining [3H]TdR autoradiography with immunoperoxidase detection of BrdU in the same sections. A single peak in the appearance of double-labelled cells was seen at 44 h, if [3H]TdR was given at 1200 h; following [3H]TdR at 0300 h, a peak of double labelling was seen at 48 h with the possibility of smaller peaks at 24 h and 36 h. These results show that the 24 h periodicity in LI in this tissue is associated with a predominant cell cycle duration of 44-48 h, but that a few cells cycle more quickly. Double labelling with [3H]TdR and BrdU provides a useful method for establishing cell cycle duration by labelling S-phase cells in successive cell cycles.     

DNA-synthesizing cells in oral epithelium have a range of cell cycle durations: evidence from double-labelling studies using tritiated thymidine and bromodeoxyuridine

Abstract Mouse tongue epithelium is characterized by a circadian variation in the number of DNA-synthesizing cells (labelling index, LI). Cells undergoing DNA synthesis were labelled with tritiated thymidine ([³H]TdR) at 0300 (peak LI) or 1200 h (low LI). The fate of these cells was assessed by injecting animals with bromodeoxyuridine (BrdU) at intervals from 12–48 h after [³H]TdR, to follow them from one cell cycle to the next. Labelling was revealed by combining [³H]TdR autoradiography with immunoperoxidase detection of BrdU in the same sections.
A single peak in the appearance of double-labelled cells was seen at 44 h, if [³H]TdR was given at 1200 h; following [3H]TdR at 0300 h, a peak of double labelling was seen at 48 h with the possibility of smaller peaks at 24 h and 36 h.
These results show that the 24 h periodicity in LI in this tissue is associated with a predominant cell cycle duration of 44–48 h, but that a few cells cycle more quickly. Double labelling with [³H]TdR and BrdU provides a useful method for establishing cell cycle duration by labelling S-phase cells in successive cell cycles.     

Effect of a stable analog of leu-enkephalin, dalargin, on the cell division of the corneal epithelium in white rats

The effect of Leu-enkephalin analog--dalargin--on the corneal epithelium proliferation has been studied in white rats. 10 microliter dalargin per 1 kg body weight were administered intraperitoneally at 8 a.m. The mitotic index (MI), DNA synthesis cell index and label intensity (LI) were determined every 4 hours over a 24-hour period. The results obtained demonstrate that dalargin stimulates DNA synthesis in cells throughout the entire period of action. MI increased only 4, 8, 12 hours after dalargin administration. Mean daily DNA synthesis cell index and MI increased 2.1-fold and 3.1-fold, respectively after dalargin administration. It is suggested that dalargin activates the cell division processes by speeding up mitosis, shortening the premitotic period, accelerating the speed of the DNA synthesis and increasing cell proliferation pool.     

Proliferative Response and Renewal of Hepatic Function Following Cocaine Administration In Mice

ABSTRACT Experiments were undertaken to investigate the hepatic, temporal and spatial sequence of events following a single injection of cocaine, a known hepatotoxin. Centrilobular necrosis was induced in male mice (DBA/2Ha) 24 hr post-injection (PI). the time course of hepatic damage was monitored by assaying microsomal cytochrome P₄₅₀ content, the activity of microsomal FAD-containing monooxygenase (FAD-M) and by determining the levels of serum glutamic pyruvic transaminase (SGPT). Kinetics of the onset of DNA synthesis were determined by autoradiography of thin liver sections and the incorporation of ³H-methyl thymidine into perchloric-acid-precipitable material. There was no increase in the labelling index (LI) and thymidine (TdR) incorporation in the first 24 hr PI. the LI rose to 14.6% and TdR incorporation showed a 5-fold increase over control values 48 hr PI. Both indices declined slightly at 72 hr PI and returned to control values by 96 hr PI. In contrast, the cytochrome P₄₅₀ content declined by 69%, the FAD-M activity dropped by 40% and the SGPT levels showed an 18-fold increase at 24 hr PI, coincident with cytological signs of necrosis. Although the patterns of recovery differed between these selected enzymes, normal values were attained by 96 hr PI. These results demonstrate that cell damage and hepatic dysfunction precede the onset of DNA synthesis and subsequent proliferation.     

Proliferative response and renewal of hepatic function following cocaine administration in mice

Experiments were undertaken to investigate the hepatic, temporal and spatial sequence of events following a single injection of cocaine, a known hepatotoxin. Centrilobular necrosis was induced in male mice (DBA/2Ha) 24 hr post-injection (PI). The time course of hepatic damage was monitored by assaying microsomal cytochrome P450 content, the activity of microsomal FAD-containing monooxygenase (FAD-M) and by determining the levels of serum glutamic pyruvic transaminase (SGPT). Kinetics of the onset of DNA synthesis were determined by autoradiography of thin liver sections and the incorporation of 3H-methyl thymidine into perchloric-acid-precipitable material. There was no increase in the labelling index (LI) and thymidine (TdR) incorporation in the first 24 hr PI. The LI rose to 14.6% and TdR incorporation showed a 5-fold increase over control values 48 hr PI. Both indices declined slightly at 72 hr PI and returned to control values by 96 hr PI. In contrast, the cytochrome P450 content declined by 69%, the FAD-M activity dropped by 40% and the SGPT levels showed an 18-fold increase at 24 hr PI, coincident with cytological signs of necrosis. Although the patterns of recovery differed between these selected enzymes, normal values were attained by 96 hr PI. These results demonstrate that cell damage and hepatic dysfunction precede the onset of DNA synthesis and subsequent proliferation.     

Spermatogonial multiplication in the Chinese hamster. IV. Search for long cycling stem cells

In the Chinese hamster, 17 days, i.e. one cycle of the seminiferous epithelium, after two injections of [3H]TdR given 24 hr apart, labelled cells were found among all types of spermatogonia, including stem cells (As). These labelled As spermatogonia derive from one or more self-renewing divisions of the stem cells that originally incorporated [3H]TdR. In the steady state, half of the divisions of the As will be self-renewing and the other half will give rise to Apr spermatogonia that will ultimately become spermatozoa. Theoretically, the labelling index (LI) after 17 days will be similar to that after 1 hr, and in this study twice as high as for the 1-hr interval since only one injection was given. However, experimental values only half that of the theoretical LI were found after 17 days. The following causes for the loss of labelled stem cells are discussed: (1) dilution of label because of division; (2) influx of unlabelled components of false pairs (i.e. newborn stem cells that still have to migrate away, mostly during G1, from their sister cells and are scored as Apr spermatogonia) between 1 hr and 17 days; (3) the existence of long- and short-cycling stem cells, probably combined with preferential differentiation of the short-cycling elements; (4) selective segregation of DNA at stem cell mitosis; and (5) irradiation death of radiosensitive labelled stem cells. As it is not impossible that factors 1, 2, 4 and 5 together account for the total loss of labelled stem cells, LI results do not provide evidence for the existence of separate classes of short- and long-cycling stem cells. The distributions of the LIs of the As, Apr and Aal spermatogonia over the stages of the epithelial cycle at 17 days are similar to those at 1 hr after injection. Hence the regulatory mechanisms that govern the stimulation and inhibition of proliferation of As that give rise to new As for the next epithelial cycle are similar to those of the As that will divide into Apr spermatogonia during the same epithelial cycle. Grain counts revealed that more [3H]TdR is incorporated into As, Apr and Aal spermatogonia that are in S phase during epithelial stages X-IV than in stages V-IX.     

Negative effect of iodide on the survival of newly divided epithelial cells in chronically stimulated rat thyroid

The aim of this work was to investigate some aspects of the thyroid epithelial cell kinetics during the iodide-induced involution of a hyperplastic goitre in the rat. Rats were made iodine-deficient for 6 months, and propylthiouracil (PTU) (0.15%) was added to the diet during the last 2 months. Thereafter, rats were refed with iodide and PTU was removed (day 0). Forty-eight hours previously, all the rats were injected with tritiated thymidine ([3H]TdR) (1 microCi/g). Some animals were killed 1 hr or 24 hr after [3H]TdR injection (i.e. on day -2 and -1, day 0 corresponding to the restoration of a normal iodine diet); the other animals were killed after different delay periods and following [3H]TdR injection. Autoradiography of thyroid sections, iodine determination of plasma iodide and protein-bound iodine (PBI), and RIA of plasma thyroid stimulatory hormone (TSH) were performed. Plasma TSH concentration was very high on day 0 of iodide refeeding (3000 +/- 330 ng/ml) and remained at this level until day 8. Plasma PBI was very low on day 0, remained so until day 4 and greatly increased on day 8. Plasma iodide was also very low on day 0, but markedly increased on day 1, then did not vary significantly until day 43 of iodine refeeding. Thyroid weight, elevated on day 0, decreased relatively quickly until day 30, then more slowly until day 73. The [3H]TdR labelling index (LI) of the thyroid epithelial cells (TEC) was high on day 0 (56 +/- 3 labelled cells/10,000 cells), and 24 hr thereafter increased to 104 +/- 3, by division of the labelled cells. On day 1 of iodine refeeding, the LI had abruptly decreased to about half this value and then remained stable for 3 more days. Between day 4 and day 16, a progressive decline in the LI, (by about 3-4 per day), was observed. The LI showed no further modification, up to day 73, the longest period investigated. The decrease in LI occurred without any significant changes in the labelling intensity (grain count) of the remaining labelled cells between day 1 and 16, this indicates that no cell division took place during this period. The data are therefore interpreted as showing a biphasic elimination after iodide refeeding, of cells that were actively proliferating during the goitrous state.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of pulse labelling with tritiated thymidine on the circadian rhythmicity in epidermal cell-cycle distribution in mice

The influence of pulse labelling with 50 microCi tritiated thymidine ( [3H]TdR) (2 microCi/g) on epidermal cell-cycle distribution in mice was investigated. Animals were injected intraperitoneally with the radioactive tracer or with saline at 08.00 hours, and groups of animals were sacrificed at intervals during the following 32 hr. Epidermal basal cells were isolated from the back skin of the animals and prepared for DNA flow cytometry, and the proportions of cells in the S and G2 phases of the cell cycle were estimated from the obtained DNA frequency distributions. The proportions of mitoses among basal cells were determined in histological sections from the same animals, as were the numbers of [3H]TdR-labelled cells per microscopic field by means of autoradiography. The results showed that the [3H]TdR activity did not affect the pattern of circadian rhythms in the proportions of cells in S, G2 and M phase during the first 32 hr after the injection. The number of labelled cells per vision field was approximately doubled between 8 and 12 hr after tracer injection, indicating an unperturbed cell-cycle progression of the labelled cohort. In agreement with previous reports, an increase in the mitotic index was seen during the first 2 hr. These data are in agreement with the assumption that 50 microCi [3H]TdR given as a pulse does not perturb cell-cycle progression in mouse epidermis in a way that invalidates percentage labelled mitosis (PLM) and double-labelling experiments.     

Cell population kinetics in pig epidermis: further studies

The cell population kinetics of the epidermis were studied in 4-month-old pigs. Mitotic figures were confined to the basal cell (L1) and the first suprabasal cell layer (L2). The mitotic index (MI) was 0.17 +/- 0.04% for L1 and 0.08 +/- 0.03% for L2. Labelled nuclei were distributed throughout the viable epidermis, the majority (79.1 +/- 1.1%) were in L1 with 19.5 +/- 1.2% in L2. The labelling indices (LI) in layers L1 and L2 were 7.1 +/- 0.4% and 3.4 +/- 0.1%, respectively. After labelling with two injections of tritiated thymidine [3H]TdR separated by 90 min, the LI increased to 8.2 +/- 0.3% in L1 and to 4.0 +/- 0.2% in L2. This increased labelling confirmed that cell proliferation occurs in both layers, L1 and L2, of the epidermis. The cell production rate (K) in L1 and L2 had an upper limit of 10.7 +/- 1.0 and 6.2 +/- 1.8 cells per 1000 cells per hour respectively. The cell flow rate per hour (cell flux), into and out of the DNA synthesis phase (S), and the duration of DNA synthesis were determined from double-labelling studies with [3H]TdR and [14C]TdR. The cell flux into and out of S was identical and was calculated as 0.6 +/- 0.1%/hr (L1) and 0.5 +/- 0.1%/hr (L2). Values for tS varied from 8 to 10 hr. The cell turnover times (tT) were in the range 89-129 hr and 180-261 hr for L1 and L2, respectively. Log normal curves were fitted to the fraction labelled mitoses data for L1 and L2. Values for tS for cells in L1 and L2 were 9.8 hr and 11.9 hr, respectively. tG2 + 1/2tM was 7.2 hr in L1 and 9.1 hr in L2.     

An artifact in measurement of S phase initiation and its implication for the kinetics of S phase-specific enzyme activities

It is shown that the different onset of S phase as measured by autoradiography vs cumulative thymidine uptake is an artifact. We consequently propose that S phase-specific enzyme activities may accumulate a few hours prior to the actual initiation of DNA synthesis. A “pre-S” DNA synthesis that can be readily detected only by autoradiography has been proposed. Published data show that DNA synthesis in cultured animal cells is initiated approx. 2 h later when measured by cumulative incorporation of [³H]thymidine ([³H]TdR) as compared with autoradiography. We show here that the difference is in reality an artifact, owing to not taking into account both gradual, asynchronous entry of cells into S phase, as well as time-dependent accumulation of radioactivity into each cell after it has entered S phase. Combination of these two factors leads to the conclusion that [³H]TdR should be incorporated approximately as the square of time following entry of the first cell into S. Taking this into account, the two methods then are in agreement, as predicted. This argument also applies to the enzyme activities shown to increase with DNA synthesis in synchronized cultures. Such an enzyme accumulation really could begin some time earlier than indicated by conventional plots of cumulative enzyme activity vs time and may, in fact, precede the onset of S by a few hours.     

Cell population kinetics of thyroid follicular cells in infant rats

Abstract. T cell population kinetics of thyroid follicular cells in rats were studied by means of autoradiography and a statmokinetic technique. During the first fortnight after birth no significant changes in the mitotic index (MI) and labelling index (LI) were found. In the next 2 weeks a constant decrease in the number of proliferating cells occurs. In 10-day old animals 40% of the follicular cells were in the cell cycle (GF); 3.25 ± 0.77 (SEM) % in the S phase and 0.18 ± 0.04% in mitoses (MI). Day–night changes in the LI and mitotic rate (MR) indicated a peak value at 13.30 hours with a lowest value at 22.30 hours. The mean LI and MR averaged over the whole 24 hr were 3.1 ± 0.1% and 122.2 ± 18.1%, respectively. In 10-day old animals, using the fraction of labelled mitoses (FLM) method the median cell cycle time ( T _C) was 79 hr and the phase durations were T _G1—64.6 hr, T _s—8.2 hr and T _G2—5.1 hr. The decrease in the number of proliferating cells with the age of the animals is considered to be a result of both cell cycle prolongation and in growth fraction reduction.     

Quantitative study of deoxycytidine incorporation in large and small lymphocytes of the mouse

Using radioautographic smear preparations of thymocytes and mesenteric lymph node (MLN) cells labelled with three different tritiated pyrimidine deoxyribonucleosides, the incorporation of DNA precursors was studied separately on large lymphocytes and small lymphocytes. Radioautographic reaction due to generally tritiated deoxycytidine ( [G-3H]CdR) labelling in vivo in large lymphocytes was more intense than that in small lymphocytes. When mice were sacrificed 6 hr after the administration of tritiated thymidine ( [3H]TdR), small lymphocytes were labelled more heavily than large lymphocytes. However, labelling intensity with [3H]TdR in large lymphocytes was greatly enhanced by the administration of 5-fluoro-deoxyuridine, whereas in small lymphocytes labelling intensity was only fairly enhanced by the same treatment. When cells were incubated in vitro with 5-tritium labelled deoxycytidine [( 5-3H]CdR) for 10 min, there was no significant difference in labelling intensities between large and small lymphocytes. In the case of [G-3H]CdR incorporation, the labelling intensity in large lymphocytes was found to be significantly stronger than that in small lymphocytes. Large as well as small lymphocytes incorporated [3H]TdR very well in vitro. However, addition of 5 X 0 X 10(-5) M of non-radioactive CdR to the medium greatly decreased the incorporation of [3H]TdR by large lymphocytes, whereas the effect of non-radioactive CdR in small lymphocytes was not so marked as that in large lymphocytes. Furthermore, the [3H]TdR-labelling percentages were decreased at the same rate by the addition of non-radioactive CdR in both large and small lymphocytes. These results indicate that large lymphocytes and a proportion of small lymphocytes have a strong tendency to convert CdR to thymidine mono-phosphate, which is utilized for DNA synthesis, whereas this ability is relatively weak in the rest of small lymphocytes. Thus, it is probably that this metabolic ability changes during the transition of the large lymphocyte to the small lymphocyte.     

Proceedings: Heterochromatin and chromosome aberrations: a comparative study of normal and tumour cells of mouse with various distributions of heterochromatin.

Seedlings of Crepis capillaris were irradiated after pulse-labelling with tritiated thymidine ([³H]TdR), and both chromosomal aberrations and presence of silver grains were recorded in the same metaphase cells at various intervals throughout the whole mitotic cycle. The following results were obtained: (a) irradiated roots were homogeneous with respect to the number of aberrations, and heterogenous with respect to labelling index (LI); (b) time-effect curves for labelled (L) and unlabelled (U) cells showed no significant difference from one another; (c) no significant quantitative difference of aberration spectra produced in S and G₂ stages was found. These results support the view that the major factor which determines both quantitative and qualitative variation in the production of chromosomal aberrations by radiation is the time lapse between irradiation and fixation rather than relation of the time of irradiation to the time of DNA synthesis. In addition, it was found that labelling with [³H]TdR modifies the effect of radiation on chromosomes.     

A long-lived thymidine pool in epithelial stem cells

Abstract. The labelling index (LI) of the individual basal cell positions of the anterior column of mouse tongue filiform papillae was assessed with time after an injection of [³H]TdR at 12.00 hours (the minimum point in the circadian LI rhythm). An initial doubling of the LI in the stem cell zone due to cell division was followed by a second rise of 14–16% 16 hr after injection and this occurred even in the presence of vincristine. Although the uptake of [³H]TdR and the initial LI doubling were largely prevented by a preceding injection of hydroxyurea, the 14–16% LI rise was still observed. The possible explanations are discussed, the favoured one being that an average of one of the six or seven cells (the stem cell) in each stem cell zone can store [³H]TdR in a long-lived precursor pool for at least 16 hr before being utilized for DNA synthesis. This complements previously published work which suggested that one cell in each stem cell zone may selectively segregate DNA at mitosis.     

Effects of Pulse Labelling With Tritiated Thymidine On the Circadian Rhythmicity In Epidermal Cell-Cycle Distribution In Mice

The influence of pulse labelling with 50 °Ci tritiated thymidine ([³H]TdR) (2 μCi/g) on epidermal cell-cycle distribution in mice was investigated. Animals were injected intraperitoneally with the radioactive tracer or with saline at 08.00 hours, and groups of animals were sacrificed at intervals during the following 32 hr. Epidermal basal cells were isolated from the back skin of the animals and prepared for DNA flow cytometry, and the proportions of cells in the S and G₂ phases of the cell cycle were estimated from the obtained DNA frequency distributions. the proportions of mitoses among basal cells were determined in histological sections from the same animals, as were the numbers of [³H]TdR-labelled cells per microscopic field by means of autoradiography. The results showed that the [³H]TdR activity did not affect the pattern of circadian rhythms in the proportions of cells in S, G₂ and M phase during the first 32 hr after the injection. the number of labelled cells per vision field was approximately doubled between 8 and 12 hr after tracer injection, indicating an unperturbed cell-cycle progression of the labelled cohort. In agreement with previous reports, an increase in the mitotic index was seen during the first 2 hr. These data are in agreement with the assumption that 50 °Ci [³H]TdR given as a pulse does not perturb cell-cycle progression in mouse epidermis in a way that invalidates percentage labelled mitosis (PLM) and double-labelling experiments.     

Unbalanced growth and cell death in HeLa S3 cultures treated with DNA synthesis inhibitors

Flow cytometry indicated that significant amounts of dsRNA were accumulated in HeLa S3 cells blocked at or near G₁/S boundary by hydroxyurea (HU) or excess thymidine (TdR). The dsRNA/DNA ratio increased in these cells in a manner characteristic of unbalanced cell growth. In HU-treated cells, dsRNA content was maximal 16 hours after addition of the drug and did not change significantly during the next 24 hours. The DNA content in blocked cells increased by 10%. Cell viability assessed by colony formation in soft agar decreased exponentially in HU-treated cultures after 16 hours of incubation. Correlation between loss of cell viability and rate of cell proliferation after removal of HU was observed, as determined by cell count and analysis of cell cycle progression. In TdR-treated cultures cells slowly progressed into mid S-phase during 40 hours and dsRNA accumulation continued during this period. Cell viability was not significantly affected by treatment with excess TdR, indicating that unbalanced growth per se, as measured by dsRNA accumulation, is not lethal for the cells. After reversal of DNA synthesis inhibition by removal of the drug, cells treated with HU for 16 hours or TdR for 16–24 hours promptly progressed through the cell cycle. This progression was accompanied by accumulation of significant amounts of dsRNA. As a result, cells in G₂ phase had a very high dsRNA content leading to retention of the unbalanced condition (increased dsRNA/DNA ratio) in the daughter cells. It is suggested that dsRNA accumulation in the cell is controlled to a certain degree by cell progression through the S phase. This type of control, evidently, was reflected in limited dsRNA accumulation in the cells blocked at or near G₁/S border, in continuous dsRNA accumulation in the cells slowly progressing through S phase, and in accumulation of large amounts of dsRNA after renewal of progression through the S phase.     

Effect of amino acid deprivation on DNA synthesis in BHK-21/C13 cells

Removal of serum from BHK-21/C13 cells in culture results in a decline in thymidine incorporation extending over five days. Additional removal of any of several amino acids results in a rapid decrease in incorporation of thymidine to negligible levels by 24 hours. Replacement by complete medium then provokes a synchronous wave of DNA synthesis after only ten hours with DNA synthesis first increased at six hours. Starvation for glutamine results in a rapid decline in protein synthesis over the 24 hour period when DNA synthesis is falling. However, there is considerable degradation of total protein during this period, and RNA degradation is also greatly increased. Concurrently, synthesis of RNA falls to less than 10% of that in control cells.     

Double labelling of cells with tritiated thymidine and bromodeoxyuridine reveals a circadian rhythm-dependent variation in duration of DNA synthesis and S phase flux rates in rodent oral epithelium

Abstract. We describe a double labelling method for estimating the duration of DNA synthesis (Ts) and the flux of cells into and from the S phase of the cell cycle, based on labelling with tritiated thymidine ([³H]TdR) followed by bromodeoxyuridine (BrdU) and combining immunohistological detection of BrdU with conventional autoradiography. In practice, the change in size of a window of double labelled cells occurs as the time interval between the two labels increases. In mouse tongue epithelium there is a marked circadian variation in the number of cells in DNA synthesis. From 0900 to 1500 h this labelling index (LI) falls, but from 2100 to 0300 h it increases. Our results show that the circadian decrease in LI is associated with a short Ts (5·8 ± 0·3 h), a high S phase efflux and an initially low influx of cells from G_: into S. Conversely, the rising circadian LI is associated with a longer Ts (9.4 ± 0.1 h), an initially low efflux and a moderate to high influx. Two time-points exist on the circadian LI curve when influx and efflux rates change abruptly. At 0100 h the efflux rate rises from low (5 cells %/h) to high (15–16 cells %/h) and simultaneously the influx rate changes from high to low. Similarly at 1300–1400 h, efflux rate falls from high (19–20 cells %/h) to low (4–8 cells %/h) values and influx rates change from low to high. This double labelling method has revealed that the duration of DNA synthesis varies across the circadian cycle, as do influx and efflux values which generally fall within a discrete range of high or low values. The timing of the changes in flux suggests the presence of two 'control' points on the circadian LI cycle that were previously unrecognized.     

Double labelling of cells with tritiated thymidine and bromodeoxyuridine reveals a circadian rhythm-dependent variation in duration of DNA synthesis and S phase flux rates in rodent oral epithelium

We describe a double labelling method for estimating the duration of DNA synthesis (Ts) and the flux of cells into and from the S phase of the cell cycle, based on labelling with tritiated thymidine [( 3H]TdR) followed by bromodeoxyuridine (BrdU) and combining immunohistological detection of BrdU with conventional autoradiography. In practice, the change in size of a window of double labelled cells occurs as the time interval between the two labels increases. In mouse tongue epithelium there is a marked circadian variation in the number of cells in DNA synthesis. From 0900 to 1500 h this labelling index (LI) falls, but from 2100 to 0300 h it increases. Our results show that the circadian decrease in LI is associated with a short Ts (5.8 +/- 0.3 h), a high S phase efflux and an initially low influx of cells from G1 into S. Conversely, the rising circadian LI is associated with a longer Ts (9.4 +/- 0.1 h), an initially low efflux and a moderate to high influx. Two time-points exist on the circadian LI curve when influx and efflux rates change abruptly. At 0100 h the efflux rate rises from low (5 cells %/h) to high (15-16 cells %/h) and simultaneously the influx rate changes from high to low. Similarly at 1300-1400 h, efflux rate falls from high (19-20 cells %/h) to low (4-8 cells %/h) values and influx rates change from low to high. This double labelling method has revealed that the duration of DNA synthesis varies across the circadian cycle, as do influx and efflux values which generally fall within a discrete range of high or low values. The timing of the changes in flux suggests the presence of two 'control' points on the circadian LI cycle that were previously unrecognized.