G2 sub-population in mouse liver induced into mitosis by lead acetate

A single intracardiac dose of lead acetate (40 microgram lead/g body weight) induced a 25-fold increase in mitosis of mouse hepatocytes 5 hr after injection, as determined by autoradiography. The prompt appearance of a mitotic wave and the relatively large number of mitoses suggest that the mitotic cells were derived from a hepatocyte sub-population arrested in the G2 phase. The injection of lead also stimulated a small increase in labeled hepatocytes within 6 hr. Analysis of grain counts gave no evidence for unscheduled DNA synthesis. The incremental labeled cells may have originated from a small fraction of the G1 population that was ready to enter the S phase without the usual pre-synthetic delay.     

Effects of Hydroxyurea On the Mitotic Activity and the G2 Phase In Hairless Mouse Epidermis

Hairless mice were given 5 mg hydroxyurea (HU) intraperitoneally (i.p.) followed by 0.15 mg Colcemid® at various times after HU. the animals were killed at 2 and 4 hr after Colcemid, the epidermal mitotic counts in dorsal skin were determined and the mitotic rates calculated. These were compared with the normal mitotic rates, and the ratios between the results from HU-treated and -untreated animals were calculated. Hydroxyurea caused a considerable reduction in the mitotic rate with a trough at 6 hr, followed by a wave of increased mitotic rate with a peak at 14 hr, followed by a secondary drop at 20 hr, and then a return to normal. Another group of mice were given HU only, and the fraction of epidermal cells in G₂ was measured by flow cytometry. From these animals, without previous injection of Colcemid, we also determined the mitotic counts and calculated the mitotic durations. Cells piled up in G₂ for the first 6 hr after HU injection, then the G₂ compartment was emptied. the results are discussed in relation to previous results from this department showing the effect of the same dose of HU on DNA synthesis in the same mouse strain. It is concluded that HU not only blocks or retards DNA synthesis in epidermal cells, but also affects the movement of cells through G₂ and M. the cell kinetic effects of HU thus seem to be very complex.     

Spatiotemporal changes in Ha-ras p21 expression through the hepatocyte cell cycle during liver regeneration

The protein product of the ras oncogene, Ha-ras (p21), is thought to be an important regulator of cell growth. The cytoplasmic relocalization of p21 in the cell during the cell cycle suggests a transient signaling role for this protein in association with its signal transduction function. Because of the importance of this role we examined spatial patterns in vivo of p21 expression at the protein and mRNA levels in hepatocytes during compensatory growth in rat liver following partial hepatectomy. A low level of p21 was immunolocalized on the cytoplasmic membrane of nonregenerating hepatocytes. The level of hepatic p21 increased significantly and without spatial restriction within the liver from 36 to 60 hr after partial hepatectomy (PH). p21 was localized in the cytoplasm of dividing hepatocytes and on the hepatic cytoplasmic membrane. The elevated p21 level decreased and was found mainly on hepatocyte plasma membranes by 96 hr after PH. Immunogold electron microscopy showed p21 localized over mitochondrial membranes and nuclei in nondividing regenerating hepatocytes. Approximately 50% of nonregenerating hepatocytes show nuclear localization of p21. This percentage changes with time following PH. The decrease in nuclear localization was accompanied with an increase in the low number of hepatocytes which demonstrated cytoplasmic localization in nondividing hepatocytes in regenerating liver. Flow cytometric analysis revealed a significant increase of p21 at 36 hr after PH which was 12 hr after the initial induction of ras mRNA. ras mRNA level increased 1.5-fold at 24 hr after PH and a maximum twofold induction was observed at 48 hr. Cell-cycle analysis of regenerating hepatocytes indicated a synchronized first peak of cell division 36–40 hr after PH. Dual parameter flow cytometry revealed that the level of p21 in hepatocytes in S phase and G₂/M phase of the cell cycle was significantly higher than that in G₀/G₁ phase during regeneration. These findings suggest that p21 is important for the progression of regenerating hepatocytes to S phase and then to G₂/M phase.     

Variation In G1 Transit Time Relative to the Cycloheximide and Actinomycin D Drug Restriction Points

The transit time distribution at various points in the cell cycle of synchronized Chinese hamster ovary cells was determined from the mitotic index, [³H]thymidine labeling index and increase in cell number monitored at regular intervals after mitotic selection. Variation in G₁ transit time compared with that for the total cell cycle indicates that variation in cell cycle transit time occurs mainly during G₁ phase. the cycloheximide (5.0 μg/ml) and actinomycin D (3.0 μg/ml) restriction points occur 0.2 and 1.7 hr prior to entry into S phase, respectively. the transit time distributions are further characterized by the moments of the distributions. the variance (2nd moment about the mean) of the transit time distribution at the actinomycin D restriction point is similar to the variance of the transit time distribution at the G₁/S border, thus variation in cell cycle transit time originates earlier than 1.7 hr prior to entry into S phase (i.e., the first 3/4 of G₁). If G₁ transit time variability and cell cycle control are related, then the results presented here indicate that the major regulatory events do not occur during late G₁ phase.     

EFFECT OF METHOTREXATE ON THE CELL CYCLE OF L1210 LEUKEMIA

The influence of methotrexate (MTX) on the proliferative activity of cells in different phases of cell cycle has been studied. MTX (5 mg/kg) was injected i.p. 3 days after the inoculation of 5 × 10⁶ leukemia cells into F₁ (DBA × C57 BL) mice. It was shown that MTX causes degeneration of cells, being in G₁- as well as in S-phase at the time of drug injection. Incorporation of ³H-TdR was suppressed for a period ranging from 2 to 12 hr after MTX administration, which is demonstrated by the decrease in the number of grains per cell. The number of cells labeled after ³H-TdR injection was also sharply decreased during this period. For a period of 3 until 15 hr after MTX administration the mitotic index decreased significantly as a result of inhibition of DNA synthesis. The blocking of the G₁-S transition was evident during 4 hr after MTX. Thereafter the G₁-S transition proceeds at a rate which is practically equal to that for nontreated controls. MTX did not inhibit transition to mitosis of cells being in G₂-phase and in a very late S-phase at the time of drug injection. The sensitivity of G₁-cells to the cytocidal effect of MTX shows that for L1210 leukemia cells MTX can be classified as a cycle-specific drug killing both G₁ and S-cells rather than S-phase specific agent with self-limitation.     

EXPERIMENTAL CONTROL OF DNA SYNTHESIZING AND DIVIDING CELLS IN EXCISED ROOT TIPS OF PISUM

The number of dividing and DNA-synthesizing cells in excised pea roots can be regulated by eliminating the carbohydrate normally supplied in the culture medium. When the excised roots were allowed to remain for 24 hr in a medium lacking carbohydrate, the number of mitotic figures and tritiated thymidine (H³-T) labeled cells was reduced almost to zero. After an additional 24 hr in the incomplete culture medium, 15% of the interphase cells were H³-T labeled, the percentage of the cells that were dividing never exceeded 1.4, and 30% of these were H³-T labeled. When the roots remained in the deficient medium for 72 hr, neither cell division nor cells synthesizing DNA were observed. Upon addition of 2% sucrose, cell division and DNA synthesis were resumed in the roots that were maintained for 24 or 72 hr without an exogenous carbohydrate supply. It has been hypothesized that some proliferative systems consist of two cellular subpopulations which selectively stop or remain in either the pre-DNA synthetic (G₁) or post-DNA synthetic (G₂) periods of the mitotic cycle. The addition of sucrose, H³-T, and 5-aminouracil to the medium, after the roots had been maintained for 24 hr without a carbohydrate, indicated that most of the proliferative cells in the roots had accumulated in either G₁, a quasi-G₁ condition, i.e., DNA synthesis stopped sometime before completion, or G₂ periods of interphase; the majority, however, were in G₁ or quasi-G₁ conditions. The results suggested that DNA synthesis (S period) and mitosis or the onset of these processes have the highest metabolic requirements in the mitotic cycle and that G₁ and G₂ were the most probable states for proliferative cells in a meristem with a low metabolic level.     

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.     

STUDIES ON THE REGULATION OF LYMPHOCYTE PRODUCTION IN THE MURINE THYMUS AND SOME EFFECTS OF A CRUDE THYMUS EXTRACT

Cell proliferation in the murine thymus was studied in vivo under normal conditions and from 0 to 24 hr after a single injection of a water-soluble extract from mouse thymus, mouse spleen, and mouse skin. The thymus extract reduced during the first 24 hr the mitotic activity 40%; the spleen extract had a weaker inhibitory effect. The skin extract had no such effect. The thymus extract and spleen extract inhibited the flux of cells into the S phase 0–8 hr after the injection of the extract. Initial labelling index was also reduced in this period. Eight hours after injection of the thymus or spleen extracts the inhibited cells initiated DNA synthesis. The rate of progression of blast cells through the cell cycle was normal 24 hr after the injection of the extracts. It was deduced from the analysis that the thymus extract inhibits processes triggering Go/Gi cells into DNA synthesis, the inhibition of G₂ efflux being of minor importance. Finally a model for the regulation of proliferating thymic blast cells and the emigration of small lymphocytes from the thymus is proposed.     

CELL CYCLE TIME DETERMINATIONS BASED ON LIQUID SCINTILLATION COUNTING OF 3H-TdR LABELED MITOTIC CELLS

Following a 10 min pulse labeling with ³H-TdR, flasks of asynchronous monolayer cultures of Chinese hamster ovary cells were subjected to mitotic selection at 2 hr intervals. The mitotic index of the selected populations was always greater than 90%. Counts per min per cell obtained by liquid scintillation counting were plotted versus time after the pulse label. Comparisons were made between cycle times obtained by the mitotic-scintillation counting method and by the standard per cent labeled mitosis technique. The resulting curves were used for calculations of the cell cycle times and the lengths of G₁, S, G₂ and M phases of the cell cycle. There was less than 2% difference in the cell cycle times obtained using the scintillation method as compared to times calculated from autoradiographic data obtained from individual petri dishes. The mitotic-scintillation counting technique is simple, accurate and rapid and allows the calculation of the cell kinetics parameters within 1 hr of the end of the experiment.     

EFFECT OF CYCLOHEXIMIDE ON THE CELL CYCLE OF THE CRYPTS OF THE SMALL INTESTINE OF THE RAT

A single injection of 1.5 mg/kg of cycloheximide induces a complete disappearance of mitotic activity in rat intestinal crypts within 1.5–2 hr. No significant necrosis of crypt cells is observed even though this phenomenon is accompanied by a marked decrease in uptake of labeled precursors into protein and DNA. Mitoses reappear 6 hr after injection and recovery then follows a cyclic pattern over a period equivalent to one cell cycle, thereby reflecting at least a partial synchronization of cell division. Concurrent use of colchicine, an agent known to induce metaphase arrest, has demonstrated that cycloheximide, while having no apparent effect on cells already in division, prevents the entrance of new cells into visible mitosis. Analysis of the cell cycle suggests that one block initiated by cycloheximide occurs in G₂, presumably as the result of an interference with the formation of protein(s) required for the normal progression of cells from this phase of the cycle into mitosis.     

Autoradiographic studies on the incorporation of3H-amino acids into chromosomes during the mitotic cell cycle ofHaplopappus gracilis

The synthesis of chromosomal proteins and the incorporation of labelled proteins into chromosomes in the mitotic cell cycle ofHaplopappus gracilis, 2n=4, were traced autoradiographically with³H-arginine,³H-lysine, and³H-tryptophane. The duration of the mitotic cell cycle in the root tip cells was determined by³H-thymidine autoradiography and was measured to be 13.0 hr (G₁ 1.3 hr, S 6.5 hr, G₂ 3.8 hr and M 1.4 hr).³H-arginine labelled proteins which were synthesized at S and G₂ were found to be incorporated into chromosomes to a greater extent than proteins which were synthesized either at G₁, at the transition phase from late S to early G₂, or at the mitotic phase. Such varied incorporation was also found in³H-lysine labelled proteins, but not in³H-tryptophane labelled proteins. These findings indicate that the chromosomal proteins are synthesized mainly at S and G₂. Some of the³H-arginine labelled proteins which were synthesized during the first mitotic cell cycle, were found to be incorporated into the chromosomes of the second mitotic cell cycle. The incorporation of the proteins synthesized at one stage of the mitotic cell cycle was found to occur locally in some regions of the chromosomes, while the pattern of incorporation was observed to be similar between euchromatic and heterochromatic regions.     

REGENERATIVE PROLIFERATION OF MOUSE EPIDERMAL CELLS FOLLOWING ADHESIVE TAPE STRIPPING

The proliferating cells of mouse epidermis (basal cells) can be separated from the non-proliferating cells (differentiating cells) (Laerum, 1969) and brought into a mono-disperse suspension. This makes it possible to determine the cell cycle distributions (e.g. the relative number of cells in the G^ S and (G₂+ M) phases of the cell cycle) of the basal cell population by means of micro-flow fluorometry. To study the regenerative cell proliferation in epidermis in more detail, changes in cell cycle distributions were observed by means of micro-flow fluorometry during the first 48 hr following adhesive tape stripping. ³H-TdR uptake (LI and grain count distribution) and mitotic rate (colcemid method) were also observed. An initial accumulation of G₂ cells was observed 2 hr after stripping, followed by a subsequent decrease to less than half the control level. This was followed by an increase of cells entering mitosis from an initial depression to a first peak between 5 and 9 hr which could be satisfactorily explained by the changes in the G₂ pool. After an initial depression of the S phase parameters, three peaks with intervals of about 12 hr followed. The cells in these peaks could be followed as cohorts through the G₂ phase and mitosis, indicating a partial synchrony of cell cycle passage, with a shortening of the mean generation time of basal cells from 83-3 hr to about 12 hr. The oscillations of the proportion of cells in G₂ phase indicated a rapid passage through this cell cycle phase. The S phase duration was within the normal range but showed a moderate decrease and the Gj phase duration was decreased to a minimum. In rapidly proliferating epidermis there was a good correlation between change in the number of labelled cells and cells with S phase DNA content. This shows that micro-flow fluorometry is a rapid method for the study of cell kinetics in a perturbed cell system in vivo.     

DNA synthesis and the cell cycle in cultures of normal and mutant (mdg/mdg) embryonic mouse muscle cells.

The relationship between cell fusion, DNA synthesis and the cell cycle in cultured embryonic normal and dysgenic ($\text{mdg}\text{mdg}$) mouse muscle cells has been determined by autoradiography. The experimental evidence shows that the homozygous mutant myotubes form by a process of cell fusion and that nuclei within the myotubes do not synthesize DNA or undergo mitotic or amitotic division. The duration of the total cell cycle and its component phases was statistically the same in 2-day normal and mutant ($\text{mdg}\text{mdg}$) myogenic cultures with the approximate values: T, 21.5 hr; G₁, 10.5 hr; S, 7.5 hr; and G₂, 2.5 hr. In both kinds of cultures, labeled nuclei appeared in myotubes 15–16 hr after mononucleated cells were exposed to [³H]thymidine, and the rate of incorporation of labeled nuclei into multinucleated muscle cells was comparable in control and dysgenic cultures. Thus, homozygous $\text{mdg}\text{mdg}$ muscle cells in culture are similar to control cells with respect to their mechanism of myotube formation and the coordinate regulation of DNA synthesis and the cell cycle during myogenesis.     

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.     

Importance of cell aggregation for expression of liver functions and regeneration demonstrated with primary cultured hepatocytes

Adult rat hepatocytes aggregated to form floating multicellular spheroids when cultured in Primaria dishes, which have a positively charged surface, in serum-free Williams' medium E (WE) supplemented with insulin and epidermal growth factor (EGF). These hormones were essential for maintenance of the spheroids, whereas the size of the spheroids depended on the inoculum cell density. The spheroids retained in vivo levels of expressions of albumin and glucokinase and synthesized scarcely any DNA even in the presence of insulin and EGF. On transfer to type I collagen-coated dishes, the spheroids gradually disaggregated and the cells formed monolayers, in which the expressions of albumin and glucokinase were suppressed and DNA synthesis and hexokinase activity were increased. DNA synthesis of hepatocytes in monolayer culture was maximal 24 hr after transfer of the spheroids, ～80% of the hepatocyte nuclei were labelled with bromodeoxyuridine during culture for 48 hr, and the mitotic index was ～70% after 60 hr. These results suggest that, in spheroids, hepatocytes remained in the G₀ phase, but that when they formed monolayers, they progressed to the G₁ phase and proceeded through the cell cycle in the presence of insulin and EGF. This work shows that the cell cycle of hepatocytes in culture can be manipulated by providing conditions for quiescence as spheroids or growth as monolayers and that the shape of hepatocytes is important for regulating their growth and liver-specific functions. © 1993 Wiley-Liss, Inc.     

Cell Synchrony Techniques. I. A Comparison of Methods

Abstract Selected cell synchrony techniques, as applied to asynchronous populations of Chinese hamster ovary (CHO) cells, have been compared. Aliquots from the same culture of exponentially growing cells were synchronized using mitotic selection, mitotic selection and hydroxyurea block, centrifugal elutriation, or an EPICS V cell sorter. Sorting of cells was achieved after staining cells with Hoechst 33258. After synchronization by the various methods the relative distribution of cells in G₁ S, or G₂+ M phases of the cell cycle was determined by flow cytometry. Fractions of synchronized cells obtained from each method were replated and allowed to progress through a second cell cycle. Mitotic selection gave rise to relatively pure and unperturbed early G₁ phase cells. While cell synchrony rapidly dispersed with time, cells progressed through the cell cycle in 12 hr. Sorting with the EPICS V on the modal G₁ peak yielded a relatively pure but heterogeneous G₁ population (i.e. early to late G₁). Again, synchrony dispersed with time, but cell-cycle progression required 14 hr. With centrifugal elutriation, several different cell populations synchronized throughout the cell cycle could be rapidly obtained with a purity comparable to mitotic selection and cell sorting. It was concluded that, either alone or in combination with blocking agents such as hydroxyurea, elutriation and mitotic selection were both excellent methods for synchronizing CHO cells. Cell sorting exhibited limitations in sample size and time required for synchronizing CHO cells. Its major advantage would be its ability to isolate cell populations unique with respect to selected cellular parameters.     

CIRCADIAN RHYTHMS IN MOUSE EPIDERMAL BASAL CELL PROLIFERATION

Several kinetic parameters of basal cell proliferation in hairless mouse epidermis were studied, and all parameters clearly showed circadian fluctuations during two successive 24 hr periods. Mitotic indices and the mitotic rate were studied in histological sections; the proportions of cells with S and G₂ phase DNA content were measured by flow cytometry of isolated basal cells, and the [³H]TdR labelling indices and grain densities were determined by autoradiography in smears from basal cell suspensions. The influx and efflux of cells from each cell cycle phase were calculated from sinusoidal curves adapted to the cell kinetic findings and the phase durations were determined. A peak of cells in S phase was observed around midnight, and a cohort of partially synchronized cells passed from the S phase to the G₂ phase and traversed the G₂ phase and mitosis in the early morning. The fluctuations in the influx of cells into the S phase were small compared with the variations in efflux from the S phase and the flux through the subsequent cell cycle phases. The resulting delay in cell cycle traverse through S phase before midnight could well account for the accumulation of cells in S phase and, therefore, also the subsequent partial synchrony of cell cycle traverse through the G₂ phase and mitosis. Circadian variations in the duration of the S phase, the G₂ phase and mitosis were clearly demonstrated.     

THE INFLUENCE OF INJECTED TRITIATED THYMIDINE ON THE MITOTIC CIRCADIAN RHYTHM IN THE EPITHELIUM OF THE HAMSTER CHEEK POUCH*

The initial effect of an injection of TdR-5-³H (1 μCi/g body weight; 6 Ci/mmol) in the cheek pouch epithelium of the Syrian hamster is an increase in the mitotic index. The increase is observed 1–5 hr after injection, depending upon the time of day when the injection is given, and is followed by compensatory variations in mitotic index. This deviation from the normal circadian rhythm in the mitotic index appears to depend on the fraction of G₂-cells at the time of injection. The main effect is a shortening of t_g2. No effect is observed after injection of non-radioactive TdR or isotonic saline. The results of the present experiment emphasize that unexpected results may be obtained when using mitotic indices from animals labelled with ³H-TdR, as well as the risks of using the PLM-method in a partially synchronized system.     

5-AMINOURACIL TREATMENT : A Method for Estimating G2

Treatment of Vicia faba lateral roots with a range of concentrations of 5-aminouracil (5-AU) indicate that cells are stopped at a particular point in interphase. The timing of the fall in mitotic index suggests that cells are held at the S - G₂ transition. When cells are held at this point, treatments with 5-AU can be used to estimate the duration of G₂ + mitosis/2 of proliferating cells. Treatment with 5-AU can also be used to demonstrate the presence of subpopulations of dividing cells that differ in their G₂ duration. Using this method, 5-AU-induced inhibition, we have confirmed that in V. faba lateral roots there are two populations of dividing cells: (a) a fast-dividing population, which makes up ~85% of the proliferating cell population and has a G₂ + mitosis/2 duration of 3.3 hr, and (b) a slow-dividing population, which makes up ~15% of dividing cells and has a G₂ duration in excess of 12 hr. These estimates are similar to those obtained from percentage labeled mitosis (PLM) curves after incorporation of thymidine-³H.     

Cultured Human Tumour Cells May Be Arrested In All Stages of the Cycle During Stationary Phase: Demonstration of Quiescent Cells In G1, S and G2 Phase

Six human colon carcinoma cell lines were induced to enter stationary phase of growth by nutrient deprivation and cell crowding. Growth kinetics parameters (cell number, flow cytometric analysis of DNA distribution, and labelling and mitotic indices) were measured sequentially for all lines during the various stages of in vitro growth. Our results demonstrated that a substantial fraction of cells (9–18%) were located in G₂, phase when they changed from an exponential to a stationary mode of growth. Moreover, a large number of cells in stationary phase of growth had an S-phase DNA content, as determined by flow cytometry, but failed to incorporate radioactive DNA precursors (up to 15-fold difference). to substantiate these findings. cells in stationary phase of growth were induced to enter exponential growth by re-seeding in fresh medium at a lower density. Subsequently observed changes in DNA-compartment distribution, and in labelling and mitotic indices were those expected from cells that had been arrested at different stages of the cycle during their previous stationary phase. Thus, the non-proliferating quiescent state (Q), traditionally located ‘somewhere’ in G₁, phase, appears to be composed also of cells that can be arrested at other stages of the cycle (Q_s, and Q_G). Although the proportion of such cells is rather small, their contribution to the growth kinetics behaviour of human in vivo tumours will become apparent following ‘recruiting’ or ‘synchronizing’ clinical manoeuvres and will prevent the formation of a clear-cut wave of synchronized cells.