Circadian-Stage Dependence of Methotrexate In A Keratinized Epithelium. an in-vivo Study Using Flow Cytometry On the Hamster Cheek Pouch Epithelium

The partially synchronized cell system of the hamster cheek pouch epithelium shows a characteristic diurnal rhythm of cell proliferation. Bolus injections of methotrexate (Mtx) in both lethal (10 g/m²) and non-lethal (2 g/m²) doses were found to inhibit cell-cycle progression primarily by impairing the G₁/S transition. the results were obtained by flow cytometric DNA analysis. the inhibitory effect of Mtx manifested itself as a relative decrease of the S fraction (drug-effector phase), and was found to be dependent both on the dose and on the time of the day it was given. A bolus injection of Mtx was given either at 1200 hr (when a minimal number of cells are in S phase) or at 0200 hr (when a maximum number of cells are in S phase). the greatest cumulative decrease in S fraction was seen when the injection was given at 1200 hr. the time between injection and the effect (seen as a decrease in S fraction) was independent of the time of the Mtx injection, but seemed instead to be related to the natural diurnal period of increasing flux from G₁ to S phase (at the onset of the dark period). the main effect (the relative decrease in S fraction) was repeated during the following 24-hr period, pointing to a protracted effect of Mtx on G₁ cells. G₁ cells affected by the initial high Mtx plasma concentration seem to be responsible for the reduced influx into S phase in both the first and second 24-hr period. In earlier toxicological studies, the survival rate of hamsters was dependent on the time of injection and was highest after injection at 1200 hr. Thus maximum cytokinetic effect on epithelial cells was found at the time of the day when there was a minimum lethal effect on the animal.     

Effect of methotrexate on cells in a keratinized epithelium with an active thymidine salvage pathway assessed by autoradiography

In the partially synchronized cell system of the hamster cheek pouch epithelium, the inhibitory effect of a bolus injection of methotrexate (Mtx) (2 g/m2, injected at 1200 hr) was analysed by means of both autoradiography and flow cytometry (FCM) in a 21-hr experiment. For autoradiography [3H]TdR and [3H]UdR were used as tracers for salvage and de novo pathways of thymidylate (TMP) synthesis, respectively. For FCM no tracers were injected. The autoradiographic studies demonstrated an active TdR salvage pathway for DNA synthesis, not affected by the impaired de novo TMP synthesis. The blocked de novo TMP synthesis was partially released 7 hr after Mtx injection, but it had not totally recovered at the end of the experiment. The decrease in the fraction of S-phase cells detected about 10 hr after Mtx injection by autoradiographic labelling with [3H]TdR and by FCM was found to be caused by a decrease in the number of cells entering S phase. However, Mtx did not influence the salvage TMP synthesis rate of cells entering S phase.     

Effect of Methotrexate On Cells In A Keratinized Epithelium With an Active Thymidine Salvage Pathway Assessed By Autoradiography*

In the partially synchronized cell system of the hamster cheek pouch epithelium, the inhibitory effect of a bolus injection of methotrexate (Mtx) (2 g/m², injected at 1200 hr) was analysed by means of both autoradiography and flow cytometry (FCM) in a 21-hr experiment. For autoradiography [³H]TdR and [³H]UdR were used as tracers for salvage and de nouo pathways of thymidylate (TMP) synthesis, respectively. For FCM no tracers were injected. the autoradiographic studies demonstrated an active TdR salvage pathway for DNA synthesis, not affected by the impaired de novo TMP synthesis. the blocked de novo TMP synthesis was partially released 7 hr after Mtx injection, but it had not totally recovered at the end of the experiment. the decrease in the fraction of S-phase cells detected about 10 hr after Mtx injection by autoradiographic labelling with [³H]TdR and by FCM was found to be caused by a decrease in the number of cells entering S phase. However, Mtx did not influence the salvage TMP synthesis rate of cells entering S phase.)     

The circadian variations in the epithelial growth of the hamster cheek pouch: quantitative analysis of DNA distributions

The pronounced diurnal rhythm in DNA distributions of the hamster cheek pouch epithelium both in the S fraction and in the (G2 + M) fraction was compared with previous studies of the changes in tritiated thymidine labelling index and mitotic activity. The DNA distributions were obtained by flow cytometry after ultrasonic disaggregation of the isolated epithelium into a suspension of single nuclei. The DNA distributions were analysed with the computer program of J. Fried (1976) and by planimetry. The S fraction was higher than the autoradiographic labelling index during the whole 24 hr period. Only the computer fitted S fraction and the labelling index had the same difference between maximal and minimal values, and maxima at the same time of day. The DNA distributions showed a diurnal release of G1 cells into S phase proceeding through (G2 + M) phase and returning to G1 phase within a 24 hr period.     

Characteristics of the isoprenaline stimulated proliferative response of rat submaxillary gland.

A simple stochastic model has been developed to determine the cell cycle kinetics of the isoprenaline stimulated proliferative response in rat acinar cells. The response was measured experimentally, using 3H-TdR labelling of interphase cells and cumulative collections of mitotic cells with vincristine. The rise and fall of the fraction of labelled interphase cells and of metaphase cells is expressed by the product of the proliferative fraction and a difference of probability distributions. The probability statements of the model were formulated and then compared by an iterative fitting procedure to experimental data to obtain estimates of the model parameters. The model when fitted to the combined fraction labelled interphase (FLIW) and fraction metaphase (FMWa) waves gave a mean Gis transit time of 21-2 hr, mean Gis +S transit time of 27-0 hr, and mean Gis + S + G2 transit time of 35-8 hr for a single injection of isoprenaline, where Gis is the initiation to S phase time. When successive injections of isoprenaline were given at intervals of 24 and 28 hr the corresponding values after the third injection were 12-4 hr, 20-8 hr and 25-7 hr respectively. The variance of the Gis phase dropped from 18-1 to 1-3 while the other variances remained unchanged. The estimated proliferative fraction was 0-24 after a single injection of isoprenaline, and 0.31 after three injections of the drug. Independently determined values of the proliferative fraction, obtained from repeated 3H-TdR injections, were 0-21 and 0-36 respectively.     

Circadian effect of ACTH 1-17 on mitotic index of the corneal epithelium of BALB/C mice

The objective was to determine the effect of ACTH 1-17, an adrenocorticotropin analogue, on the mitotic index in the corneal epithelium of mice standardized in 12 hr of light alternating with 12 hr darkness. A question asked was whether the time of administration along the 24-hr time scale influenced any response found. The findings showed that ACTH 1-17 could, depending upon when it was administered, bring about a statistically significant decrease, an increase or even no such change in the mitotic index. The greatest responses found were increases, especially when ACTH 1-17 was administered during the dark span. Also the time after injection when the responses occurred varied. The greatest response recorded was at 12 hr after injection when ACTH 1-17 was given at 2 hr into the dark with a 641% and a 718% increase with a low (0.02 IU/kg) and a higher (20 IU/kg) dose, respectively. A 3-way analysis of variance supported the conclusion that the kind-of-treatment, time-of-treatment and treatment-to-kill interval (sampling time) are important factors when determining any response to ACTH 1-17 on the mitotic index.     

Cell cycle perturbation of cultured C6 glioma cells following short-term contact with a low dose of ACNU

The purpose of this study was to investigate the cell cycle perturbation of cultured C6 rat glioma cells induced by 1-(4-amino-2-methyl-5-pyrimidyl)methyl-3-(2-chloroethyl)3-nitrosourea hydrochloride (ACNU) using simultaneous flow cytometric measurements of DNA and bromodeoxyuridine (BrdU) content. A new graphic computer program permitted the quantification of cell density in hexagonal subareas and allowed the fraction of BrdU-labeled cells with mid-S phase DNA content (FLS) to be defined in a narrow window. The cell kinetic parameters such as cell cycle time (Tc) and S phase time (Ts) were estimated from a manually plotted FLS curve at 18 and 6 hr, respectively. The major effect of ACNU on the cell cycle was an accumulation of the cells in the G2M phase 12 to 24 hr posttreatment when compared to G2M traverse of untreated cells. For the two-dimensional analysis, cells were labeled with BrdU and then treated with ACNU, or treated with ACNU and then labeled with BrdU. It was concluded that the cells in the S and G2M phases at the time of ACNU administration progressed to mitosis but that the G1 phase cells accumulated in the subsequent G2M phase. Two-dimensional FCM analysis using BrdU provided a useful tool in studying cell cycle perturbation.     

Mode of estrogen action on cell proliferation in CAMA-1 cells: II. Sensitivity of G1 phase population

The mammary cancer cell line CAMA-1 synchronized at the G1/S boundary by thymidine block or at the G1/M boundary by nocodazole was used to evaluate 1) the sensitivity of a specific cell cycle phase or phases to 17 beta-estradiol (E2), 2) the effect of E2 on cell cycle kinetics, and 3) the resultant E2 effect on cell proliferation. In synchronized G1/S cells, E2-induced 3H-thymidine uptake, which indicated a newly formed S population, was observed only when E2 was added during, but not after, thymidine synchronization. Synchronized G2/M cells, enriched by Percoll gradient centrifugation to approximately 90% mitotic cells, responded to E2 added immediately following selection; the total E2-treated population traversed the cycle faster and reached S phase approximately 4 hr earlier than cells not exposed to E2. When E2 was added during the last hour of synchronization (ie, at late G2 or G2/M), or for 1 hr during mitotic cell enrichment, a mixed response occurred: a small portion had an accelerated G1 exit, while the majority of cells behaved the same as controls not incubated with E2. When E2 addition was delayed until 2 hr, 7 hr, or 12 hr following cell selection, to allow many early G1 phase cells to miss E2 exposure, the response to E2 was again mixed. When E2 was added during the 16 hr of nocodazole synchronization, when cells were largely at S or possibly at early G2, it inhibited entry into S phase. The E2-induced increase or decrease of S phase cells in the nocodazole experiments also showed corresponding changes in mitotic index and cell number. These results showed that the early G1 phase and possibly the G2/M phase are sensitive to E2 stimulation, late G1, G1/S, or G2 are refractory; the E2 stimualtion of cell proliferation is due primarily to an increased proportion of G1 cells that traverse the cell cycle and a shortened G1 period, E2 does not facilitate faster cell division; and estrogen-induced cell proliferation or G1/S transition occurs only when very early G1 phase cells are exposed to estrogen. These results are consistent with the constant transition probability hypothesis, that is, E2 alters the probability of cells entering into DNA synthesis without significantly affecting the duration of other cell cycle phases. Results from this study provide new information for further studies aimed at elucidating E2-modulated G1 events related to tumor growth.     

The Circadian Variations In the Eithelial Growth of the Hamster Cheek Pouch: Quantitative Analysis of Dna Distributions*

The pronounced diurnal rhythm in DNA distribution of the hamster check pouch epithelium both in the S fraction and in the (G₂+ M) fraction was compared with previous studies of the changes in tritiated thymidine labelling index and mitotic activity. the DNA distributions were obtained by flow cytometry after ultrasonic disaggregation of the isolated epithelium into a suspension of single nuclei. the DNA distributions were analysed with the computer program of J. Fried (1976) and by planimetry. the S fraction was higher than the autoradiographic labelling index during the whole 24 hr period. Only the computer fitted S fraction and the labelling index had the same difference between maximal and minimal values, and maxima at the same time of day. the DNA distributions showed a diurnal release of G₁ cells into S phase proceeding through (G₂+ M) phase and returning to G₁ phase within a 24 hr period.     

Diurnal Variation in Subjective and Objective Measures of Sleepiness: the Effects of Sleep Reduction and Circadian Type

In young, good sleepers the diurnal evolution of alertness was studied as a function of degree of morningness: (1) during habitual sleep routine and (2) in a 2-hr sleep reduction protocol. During habitual sleep routine, alertness was assessed using both the subjective evaluation based on Thayer's Activation Deactivation Adjective Checklist (43 subjects) and the objective measurement of sleep latency (Multiple Sleep Latency Test, MSLT). Self-alertness scored highest around midday. Later it showed a dip, then stayed on a plateau until about 2200 hr. On average, 77+ of the subjects fell asleep at the 1400 hr MSLT session while only 35.5+ did at 1000 hr and 25.8+ at 2000 hr. Morning-types (MT) and evening-types (ET) differed only during the morning: ET fell asleep more frequently at 1000 hr and 1200 hr and rated lower self-alertness on arising than did MT. Twelve subjects were given the protocol of a 2-hr sleep reduction (both in delayed bedtime and advanced rising time conditions). At 0700 hr, MT rated their alertness lower when they had only just gotten up (delayed bedtime condition) than when they had been awake for 2 hr (advanced rising time condition). In contrast, ET had the same low level of alertness at 0800 hr, independent of the time elapsed since arising. On average the advanced rising time condition affected the general pattern of alertness more than did delayed bedtime.     

20-Hydroxyecdysone accelerates the flow of cells into the G1 phase and the S phase in a male accessory gland of the mealworm pupa (Tenebrio molitor)

The cells of the bean-shaped accessory glands of mealworms proliferate through the first 7 days of the 9-day pupal stage. Immediately after larval-pupal ecdysis, 25-27% of the cells were in the G1 phase, 60-65% were in the G2 phase, and the balance were in S phase. Over the first 4 days of normal development, the S fraction gradually increased, to reach its highest level in the mid-pupa at the time of the major ecdysteroid peak (Delbecque et al., 1978). Thereafter, the S fraction declined until over 95% of the cells had accumulated in G2 on Day 8. When 0-day pupal glands were explanted into Landureau's S-20 medium for 6 days, the G1 fraction remained fairly constant (25-30%) while S and the G2 fractions fluctuated. On the first day in vitro, the G2 fraction declined and the S fraction rose. On the second day in basal media, the S fraction fell and G2 rose correspondingly until 70% of the cells reached G2 when cycling stopped on the third day. With addition of 20-hydroxyecdysone to 0-day cultures, the S fraction increased quite sharply. It remained large for all 6 days of the experiment in the continuing presence of hormone. A 1-day pulse of hormone produced a transient increase in S. We blocked cell cycling with hydroxyurea in a stathmokinetic experiment and showed that 20-hydroxyecdysone accelerated the flow of cells from the G2 phase to the G1 phase by 2.5-fold. An increase in the G1 fraction was detected within 10 hr of hormone administration and the effect was dose-dependent with an ED50 of 5 X 10(-7) M for 20-hydroxyecdysone. We conclude that 20-hydroxyecdysone acts at a control point in the G2 phase. Incubation of the glands with 20-hydroxyecdysone for only 30-60 min followed by washout stimulated the flow from G2 to G1 and the effect persisted after transfer of the tissues to hormone-free media. Dose-dependent stimulation also occurred with ponasterone A (ED50 3 X 10(-9] but not with cholesterol.     

Diurnal Variation in Subjective and Objective Measures of Sleepiness: the Effects of Sleep Reduction and Circadian Type

In young, good sleepers the diurnal evolution of alertness was studied as a function of degree of morningness: (1) during habitual sleep routine and (2) in a 2-hr sleep reduction protocol. During habitual sleep routine, alertness was assessed using both the subjective evaluation based on Thayer's Activation Deactivation Adjective Checklist (43 subjects) and the objective measurement of sleep latency (Multiple Sleep Latency Test, MSLT). Self-alertness scored highest around midday. Later it showed a dip, then stayed on a plateau until about 2200 hr. On average, 77+ of the subjects fell asleep at the 1400 hr MSLT session while only 35.5+ did at 1000 hr and 25.8+ at 2000 hr. Morning-types (MT) and evening-types (ET) differed only during the morning: ET fell asleep more frequently at 1000 hr and 1200 hr and rated lower self-alertness on arising than did MT. Twelve subjects were given the protocol of a 2-hr sleep reduction (both in delayed bedtime and advanced rising time conditions). At 0700 hr, MT rated their alertness lower when they had only just gotten up (delayed bedtime condition) than when they had been awake for 2 hr (advanced rising time condition). In contrast, ET had the same low level of alertness at 0800 hr, independent of the time elapsed since arising. On average the advanced rising time condition affected the general pattern of alertness more than did delayed bedtime.     

Circadian synchronization of hepatocyte proliferation in young rats: the role played by adrenal hormones

The circadian rhythm of hepatic cell proliferation in rats appears on the 20th day of life, when the hypothalamo-adrenal axis is mature enough for circadian activity to occur. From the 20th day to the 30th day of life, the mitotic rhythm is progressively induced by a reduction in nocturnal values, while diurnal rhythms remain unchanged. Mitotic peaks emerge at 10.00 hours. A labelling index wave occurs 8 hr before the corresponding mitotic wave, with a peak at 02.00 hours and a minimum in the evening, coincidental with the acrophase of plasma corticosterone level (activity phase). Labelled mitoses curves and metaphase accumulation after colchicine injection show that the duration of the S, G2 and M phases remain approximately constant and that the circadian variation is due to a variation in the rate of cells that enter these successive phases. During the synchronization period (from day 20 to 30), the growth fraction decreases progressively. Adrenalectomy at this time is followed by a higher cell proliferation and all rhythms disappear after 2 days. Corticosterone injected before the triggering of the rhythmic activity in 17-day-old rats immediately reduces the labelling index, while the mitotic index is decreased 10 hr later; this delay is equal to the S + G2 duration. The results are discussed. They favour the hypothesis that the circadian variation of corticosterone is responsible for the induction of a circadian variation in developmental cell proliferation by inhibition of the G1-S transition when it is higher in the evening.     

Different epidermal cell kinetic effects of hydroxyurea when injected at two different times of the day

Circadian rhythms in epidermal basal cell-cycle progression in hairless mouse skin have been repeatedly demonstrated. A dose of 10 mg/animal hydroxyurea (HU), given to inhibit DNA synthesis was injected intraperitoneally to two groups of hairless mice. One group was injected at 10.00 hours MET, when the cell-cycle progression and cell division rate are relatively high, and another group was injected at 20.00 hours, when the same variables are at minimum values. Various cell kinetic methods--[3H]TdR autoradiography, DNA flow cytometry and the stathmokinetic method (Colcemid)--were used to study HU-induced alterations in cell kinetics. Hydroxyurea (HU) immediately reduced the labelling index (LI) to less than 10% of controls when injected at both times of the day, and higher then normal values were observed 8 hr later. A subsequent decrease towards normal values was steeper in the 20.00 hours injected group. The proportion of cells with S-phase DNA content was transiently reduced in both series, but the reduction was less pronounced and control values were reached earlier in the series injected at 10.00 hours. The observed alterations in LI and fraction of cells in S phase were followed by comparable alterations in the fraction of cells in G2 and in the mitotic rate. Hence the changes in G2 and mitotic rate are easily explained as consequences of the previous perturbations in the S phase. The time-dependent differences in the cell kinetic perturbations caused by HU in the S phase may be explained by a circadian-phase-dependent action of HU on the influx and efflux of cells to and from the S phase, respectively. At 10.00 hours the efflux of cells from S is most heavily inhibited; at 20.00 hours the influx is predominantly blocked. Hence, when physiological flux is high HU mainly blocks the efflux from S, but when flux normally is low, HU mainly blocks the entrance to S. Within 20 hours after the HU injection, the cell kinetic variables had approached the unperturbed circadian pattern.     

Circadian and ultradian rhythms of proliferation in human ovarian cancer

Synchronous waves of proliferation in tumor cells taken from patients with ovarian cancer were observed using flow cytometry to measure the fraction of cells undergoing DNA replication and displaying tumor-cell-specific immunofluorescence. When saline washings of the abdominal cavity were analyzed at 2-4 hr intervals round-the-clock, the percentage of cells in the chromosome replication cycle (S + G2 percentage) showed 12-hr and often higher frequency rhythms in proliferation. These higher frequency rhythms in DNA replication show a relatively constant phase relationship to the patient's circadian clock with peak proliferation occurring most commonly at 10 a.m. to 12 noon and again at 10 p.m. This proliferation rhythm is therefore partially out of phase with the 24-hr rhythms in proliferation seen in normal cells. The findings on human cancer reveal a fundamental difference in the temporal organization of normal and tumor cell growth that should be exploited for therapeutic benefit.     

Recycling of resting cells in the JB-1 ascites tumour after treatment with 1-beta-D-arabinofuranosylcytosine (ara-C).

Resting cells in tumours present a major problem in cancer chemotherapy. In the plateau phase of grwoth of the murine JB-1 ascites tumour (i.e. 10 days after 2-5 X 10(6) cells i.p.) large fractions of non-cycling cells with G1 and G2 DNA content (Q1 and Q2 cells) are present, and the fate of these resting cells was investigated after treatment with 1-beta-D-arabinofuranosylcytosine (Ara-C).The experimental work of growth curves, percentage of labelled mitoses curves after continuous labelling with 3H-TdR, and cytophotometric determination of single-cell DNA content in unlabelled tumour cells. Treatment with an i.p. single injection of Ara-C 200 mg/kg in the plateau JB-1 tumour resulted in a significant reduction in the number of tumour cells 1 and 2 days later as compared with untreated controls, while no difference in the number of tumour cells was observed after 3 days. In tumours prelabelled with 3H-TdR 24 hr before Ara-C treatment, a significant decrease in the percentage of labelled mitoses was observed 6-8 hr later followed by a return to the initial value after 12 hr, and a new pronounced fall from 20 hr after Ara-C. The second fall in the percentage of labelled mitoses disappeared when the labelling with 3H-TdR was continued also after Ara-C treatment. Cytophotometry of unlabelled tumour cells prelabelled for 24 hr with 3H-TdR before Ara-C treatment showed 20 hr after Ara-C a pronounced decrease in the fraction of Q1 cells paralleled by an increase in the fraction of unlabelled cells with S DNA content. The results indicate recycling of resting cells first with G2 and later with G1 DNA content, which contribute to the regrowth of the tumours.     

Circadian Modification of 5-Fluorouracil-Induced Teratogenesis in Mice

Pregnant mice were treated intraperitoneally with 5-fluorouracil in a single dose of 30 mg/kg once on gestation day 11 at one of four selected times along the 24-hr time scale. All animals were kept under a lighting regimen of 12 hr (0600-1800) alternating with 12 hr of darkness. They were injected at the same circadian phase as they were mated. The developmental age of all fetuses was 264 ± 1 hr at the time of injection. Fetuses collected on day 18 showed a circadian variation in teratogenic susceptibility to 5-fluorouracil. The lethal and teratogenic risk posed by the drug were highest among animals treated at the mid-light period until the onset of darkness.     

Cell-cycle characteristics of undifferentiated and differentiating embryonal carcinoma cells

The cell-cycle parameters of undifferentiated and differentiating embryonal carcinoma cells were determined. Undifferentiated cultures of an F9 subclone, OTF9-63, had a 12-hr generation time, 10-hr S, and 2-hr G2 + M. G1 was less than 0.5 hr. In contrast, OTF9-63 cells induced to differentiate by treatment with retinoic acid had a 16.8-hr generation time, 12.5-hr S, 2-hr G2 + M, and 2.3-hr G1. Similar results were obtained with undifferentiated cultures and aggregation-induced differentiating cultures of PSA-1 cells. These data demonstrate that the undifferentiated stem cells have little or no G1, and that both G1 and S lengthen during differentiation.     

CHARACTERISTICS OF THE ISOPRENALINE STIMULATED PROLIFERATIVE RESPONSE OF RAT SUBMAXILLARY GLAND

A simple stochastic model has been developed to determine the cell cycle kinetics of the isoprenaline stimulated proliferative response in rat acinar cells. The response was measured experimentally, using ³H-TdR labelling of interphase cells and cumulative collections of mitotic cells with vincristine. The rise and fall of the fraction of labelled interphase cells and of metaphase cells is expressed by the product of the proliferative fraction and a difference of probability distributions. The probability statements of the model were formulated and then compared by an iterative fitting procedure to experimental data to obtain estimates of the model parameters. The model when fitted to the combined fraction labelled interphase (FLIW) and fraction metaphase (FMW,) waves gave a mean G_is transit time of 21-2 hr, mean G_is+ S transit time of 270 hr, and mean G_is+ S + G₂ transit time of 35-8 hr for a single injection of isoprenaline, where G_is is the initiation to S phase time. When successive injections of isoprenaline were given at intervals of 24 and 28 hr the corresponding values after the third injection were 12-4 hr, 20-8 hr and 25-7 hr respectively. The variance of the G_is phase dropped from 18-1 to 1–3 while the other variances remained unchanged. The estimated proliferative fraction was 0–24 after a single injection of isoprenaline, and 0–31 after three injections of the drug. Independently determined values of the proliferative fraction, obtained from repeated ³H-TdR injections, were 0–21 and 0–36 respectively.     

Circadian variations in influx and efflux of the S phase in a partially synchronized cell system Double-labelling with [3H]thymidine in the epithelium of the hamster cheek pouch

Abstract. By means of a double-labelling experiment, circadian variations in the kinetic parameters of the S phase of the hamster cheek pouch epithelium were studied. The evaluation of the experiment included a recently developed correction for deviations from the strict pulse interpretation of the labelling technique.
Pronounced circadian variations were found in S phase influx and efflux; the diurnal mean of both was estimated as 0.5%/hr, when based on measurements of all nucleated epithelial cells. Variations in S phase influx seem mainly responsuble for the diurnal variation in cell proliferation, although diurnal variation in DNA synthesis rate, and thus in mean transit time, was also found. The increases in LI and influx were closely correlated and related to the beginning of the dark period.
A circadian variation in cell number was also observed.