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

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

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

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

Heterogeneity in the mouse epidermal cell cycle analysed by computer simulations

Abstract. Different sets of cell kinetic data obtained over many years from hairless mouse epidermis have been simulated by a mathematical model including circadian variations. Simulating several independent sets of data with the same mathematical model strengthens the validity of the results obtained. The data simulated in this investigation were all obtained with the experimental system in a state of natural synchrony. The data include cell cycle phase distributions measured by DNA flow cytometry of isolated epidermal basal cells, fractions of tritiated thymidine ([³H]TdR) labelled cells within the cell cycle phases measured by cell sorting at intervals after [³H]TdR pulse labelling, bivariate bromodeoxyuridine (BrdUrd)/DNA data from epidermal basal cells isolated at intervals after pulse labelling with BrdUrd, mitotic rate and per cent labelled mitosis (PLM) data from histologic sections. The following main new findings were made from the simulations: the second PLM peak observed at about 35 h after pulse labelling is hardly influenced by circadian variations; the peak is mainly determined by persisting synchrony of a rapidly cycling population with a G₁-duration (T_G1) of 20 h to 30 h; and there is a highly significant population of slowly cycling G₁-cells (G_1σ). However, no significant circadian variations were found in the number of these cells.     

Laser scanning cytometer (LSC) analysis of fraction of labelled mitoses (FLM)

Abstract. In this report we describe the successful application of a novel microscope-based multiparameter laser scanning cytometer (LSC) to measure duration of different phases of cell cycle in HL-60 human leukaemic cell lines by the fraction of labelled mitoses (FLM) method. Exponentially growing cells were harvested after various time intervals following pulse-labelling with 5'-bromo-2'-deoxyuridine (BrdUrd), cytocentrifuged, fixed in ethanol, and then exposed to UV light to induce DNA strand breaks at the sites of incorporated BrdUrd. The 3'OH termini of the photolytically generated DNA strand breaks were labelled with BrdUTP in the reaction catalysed by exogenous terminal deoxynucleotidyl transferase (TdT), followed by FITC-labelled BrdUrd antibodies. DNA was counterstained with propidium iodide (PI). Due to differences in chromatin structure between the interphase and mitotic cells, the LSC identified the latter by virtue of their higher red (PI) fluorescence intensity values among all pixels over the measured cell. To confirm that the cells selected were indeed cells in mitosis, predominantly in metaphase, the recorded X-Y coordinates of selected cells were used to re-position the cell for their visual examination. From the time lapse analysis of percentage BrdUrd-labelled cells progressing through mitosis it was possible to calculate the duration of individual phases of the cell cycle. The duration of S (T_s) and G₂+ M (T_G2+M) was 8 and 3 h, respectively, and the minimal duration of G₂ (T_G2) was 2 h. The cell cycle time (T_c) estimated for the cohort of the most rapidly progressing cells was 13 h. The ability to automatically and rapidly discriminate mitotic cells combined with the possibility of their subsequent identification by image analysis makes LSC the instrument of choice for the FLM analysis.     

Intratumoral heterogeneity of S phase transition in solid tumours determined by bromodeoxyuridine labelling and flow cytometry

Abstract. Cell kinetics of human renal cell carcinomas xenotransplanted into nu/nu mice were analysed using the bromodeoxyuridine (BrdUrd) labelling method. Tumours were removed 0.5–14 h after injection of the BrdUrd solution. The tumour cells were stained with fluorescein isothiocyanate conjugated anti-BrdUrd antibodies and propidium iodide (DNA content). From the flow cytometry data the relative movement was calculated. Relative movement data of variable intervals after BrdUrd labelling were subjected to a fit procedure using log-normal distributions for S phase transition (T_s). The log-normal distributions were modified by inflation factors in order to get extremely asymmetric distributions. The best fits to the experimental data were obtained using wide asymmetric T_s distributions, indicating that progression through S phase in solid human tumours is considerably heterogeneous. This implies that the potential doubling time (T_pot) is longer than calculated from a single measured relative movement value obtained a few hours after BrdUrd labelling.     

A flow cytometric study of the effect of heat on the kinetics of cell proliferation of Chinese hamster V-79 cells

Abstract. Two methods involving labelling cells with bromodeoxyuridine (BrdUrd) have been used to study by flow cytometry the effect of hyperthermia (43°C for up to 1 h) on Chinese hamster V79 cells. One method involved the use of an antibody to BrdUrd after pulse-labelling the cells either before or at time intervals after treatment. In the second method, the cells were incubated continuously in BrdUrd after heat treatment, and the components of the cell cycle were then visualized by staining with a combination of a bis-bcnzimidazole and ethidium bromide. All three methods showed that heating at 43°C stopped DNA synthesis which, at 37°C, subsequently recovered reaching the normal rate 8–12 h later. The cells in S phase at the time of treatment then progressed to G₂ where they were further delayed. Cells heated in G₁. after the recommencement of synthesis, progressed around the cycle, albeit slower than in unheated cells. The difference between the cells in G₁ and S phases at the time of treatment may account for the greater sensitivity of S phase cells to hyperthermia.     

Normal G1/S transition and prolonged S phase within one cell cycle after seeding cells in the presence of an ornithine decarboxylase inhibitor

Abstract. We have previously found that DNA replication was affected within one cell cycle after seeding Chinese hamster ovary (CHO) cells in the presence of the polyamine biosynthesis inhibitor 2-difluoromethylornithine (DFMO). We could, however, not rule out if this was due to an effect on the G₁/S transition and/or on DNA synthesis elongation. In the present paper, we use a bromodeoxyuridine-flow cytometric method to more specifically study the G₁/S transition, the S phase length, and the progression of cells from S phase through G₂+ M and into G₁, after seeding plateau phase CHO cells at low density in the absence or presence of 5 mM DFMO. We report here that DFMO-induced polyamine depletion increased the length of the S phase within one cell cycle after seeding of CHO cells in the presence of the inhibitor. No effect on the G₁/S transition was observed until 2 days after seeding, suggesting that a DFMO-induced lengthening of the G₁ phase occurred later than the effect on S phase progression. These results imply that the G₂+ M phase was not prolonged until 2 days after seeding CHO cells in the presence of DFMO.     

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

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

A comparison of mathematical methods for the analysis of DNA histograms obtained by flow cytometry

Abstract. Twelve methods for analysing FCM-histograms were compared using the same set of data. Some of the histograms that were analysed were simulated by computer and some were taken from experiments. Simulated data were generated assuming asynchronously growing cell populations and (i) measurement coefficients of variation ( CV ) from 2 to 16%; (ii) constant measurement CV or CV 's increasing from G₁ to G₂ phase, and (iii) varying fractions of cells in each phase. Simulated data were also generated assuming synchronous cell populations in which a block in early S phase was applied and released. DNA histograms were measured for L-929 cells at various times after mitotic selection. Labelling indices were also measured for these cells at the same time.
The fractions of cells in the G₁, S, and (G₂+ M) phases were calculated by each analytical method and compared with the actual fractions used for simulation, or in case of experimental data, with autoradiographic results. Generally, all methods yielded reasonably accurate fractions of cells in each phase with relative errors in the range of 10–20%. However, most methods tended to overestimate G₁ fractions and underestimate S fractions. In addition, variations in the shape of the S phase distribution often caused considerable errors. Phase fractions were also calculated for histograms of kinetically perturbed populations, simulated as well as experimental The errors were only slightly larger than for histograms from asynchronously growing cell populations.     

Dilazep, a nucleoside transporter inhibitor, modulates cell cycle progression and DNA synthesis in rat mesangial cells in vitro

The direct effects of the nucleoside transporter inhibitor dilazep on the cell cycle of mesangial cells have not before been investigated. The purpose of this study was to elucidate whether dilazep can inhibit the proliferation of mesangial cells and how it interferes with the cell cycle of these cells. DNA histograms were used and BrdUrd uptake rate was measured by flow cytometry. There was no significant difference in the cell numbers among the untreated group and the 10⁻⁵M, 10⁻⁶M or 10⁻⁷M dilazep-treated groups at 24 h of incubation. However, at 48 and 72 h, the cell numbers in the dilazep-treated groups were significantly lower compared with that of the untreated group (P0.005). The DNA histograms of cultured rat mesangial cells at 12, 24, and 48 h of incubation with 10⁻⁵ M dilazep showed that the ratio of the S phase population in the dilazep-treated group decreased by 2.2% at 12 h, by 9.6% at 24 h, and by 18.9% at 48 h compared with the untreated group. The ratio of the G₀/G₁ phase population in the dilazep-treated group significantly increased: 6.8% at 12h (P 0.05), 13.9% at 24 h (P 0.001), and 76.5% at 48 h (P 0.001) compared with the untreated group. A flow cytometric measurement of bivariate DNA/BrdUrd distribution demonstrated that the DNA synthesis rate in the S phase decreased after 6 h (P 0.005) and 12 h (P 0.05) of incubation compared with the untreated group. These results suggest that dilazep inhibits the proliferation of cultured rat mesangial cells by suppressing the G₁/S transition by prolonging G₂/M and through decreasing the DNA synthesis rate     

Cell kinetics in mouse epidermis studied by bivariate DNA/bromodeoxyuridine and DNA/keratin flow cytometry.

Hairless mice were injected intraperitoneally with bromodeoxyuridine (Brd-Urd). Basal cells were isolated from epidermis, fixed in 70% ethanol, and prepared for bivariate BrdUrd/DNA flow cytometric (FCM) analysis. Optimum detection of incorporated BrdUrd in DNA was obtained by combining pepsin digestion and acid denaturation. The cell loss was reduced to a minimum by using phosphate-buffered saline containing Ca2+ and Mg2+ to neutralize the acid. The percentage of cells in S phase and the average uptake of BrdUrd per labelled cell in eight consecutive windows throughout the S phase were measured after pulse labelling at intervals during a 24 h period. Furthermore, the cell cycle progression of a pulse-labelled cohort of cells was followed up to 96 h after BrdUrd injection. In general the results from both experiments were in good agreement with previous data from 3H-thymidine labelling studies. The percentage of cells in S phase was highest at night and lowest in the afternoon, whereas the average uptake of BrdUrd per labelled cell showed only minor circadian variations. There were no indications that BrdUrd significantly perturbed normal epidermal growth kinetics. A cell cycle time of about 36 h was observed for the labelled cohort. Indications of heterogeneity in traverse through G1 phase were found, and the existence of slowly cycling or temporarily resting cells in G2 phase was confirmed. There was, however, no evidence of a significant population of temporarily resting cells in the S phase. Bivariate DNA/keratin FCM analysis revealed a high purity of basal cells in the suspensions and indicated that the synthesis of the differentiation-keratin K10 was turned on only in G1 phase and after the last division.     

Double labelling to obtain S phase subpopulations: application to determine cell kinetics of diploid cells in an aneuploid tumour

Abstract. We studied the cell kinetics of the murine mammary carcinoma MCa-K using iododeoxyuridine (IdUrd) and chlorodeoxyuridine (CldUrd) given at different times as independently detectable labels of S phase cells. The presence of IdUrd and CldUrd, and the amount of DNA were measured by three-colour flow cytometry making it possible to define three subpopulations within S phase and to measure the progression through the cell cycle during the time following labelling. In DNA histograms of these subpopulations, the diploid and aneuploid cells (which had a DNA index of 1.7) are essentially completely separated. From appropriate combinations of cells labelled with IdUrd only, CldUrd only, or both, it was possible to construct separate DNA distributions for the labelled diploid and aneuploid cells at the times of administration of each label. The kinetics of the diploid and aneuploid cells could be calculated for individual tumours from these two time points without having to make corrections for the presence of the second population. The diploid and aneuploid populations had indistinguishable S and G₂+ M phase durations, T_S and T_G2+M, of about 9 and 2h; however, the potential doubling time values for the aneuploid and diploid populations were 30.2 and 101.2h respectively.     

The need for dynamic methods for measuring cell cycle perturbations: a study in radiation-treated lymphoblastoid cell lines of varying p53 status

Reports on the p53-related cell cycle and apoptotic responses of EBV-transformed lymphoblastoid cell lines to DNA damage have led to some confusion. This may be due to differences in the nature of the specific p53 mutations under examination, but it can also be partly attributed to methodological and analytical problems (e.g. the inappropriate use of static DNA histograms for cell cycle analysis). Taking seven lymphoblastoid cell lines derived from both normal individuals and Li-Fraumeni Syndrome/Li-Fraumeni-Like (LFS/LFL) patients of differing p53 status, we completed a detailed study of radiation-induced cell cycle perturbations. Using BrdUrd pulse labelling and flow cytometry it was found that, regardless of p53 status, the cells did not arrest in G₁ despite all of the lines showing p53 upregulation 3 hours postirradiation. The irradiated cells did, however, show a general slowing both in S-phase entry from G₁ and in movement through S-phase. These facts would not have been apparent from the analysis of static DNA histograms. The problems with the use of static methods to assess changes in the dynamics of cell cycle progression apply not only to studies involving EBV-transformed cell lines, but also to a wide range of investigations into the molecular control of cell proliferation.     

Cell kinetic studies in mouse tongue mucosa by autoradiographic, immunohistochemical and flow cytometric techniques

Abstract. A number of techniques, including autoradiography after in vivo administration of tritiated thymidine ([³H]dT), immunohistochemistry after in vivo administration of bromodeoxyuridine (BrdUrd), and flow cytometry (FCM) with and without BrdUrd detection were compared in the epithelium of ventral mouse tongue. Investigation of the diurnal proliferative rhythm by immunohistochemical detection of incorporated BrdUrd with different primary antibodies in combination with the alkaline-phosphatase-anti-alkaline-phosphatase technique, the peroxidase-anti-perox-idase method, and an indirect method with a polyclonal peroxidase-conjugated secondary antibody yielded results similar to standard autoradiography. Preparation of single cell suspensions for flow cytometry was not successful. A maximum yield of about 8.5% of the original cell number was achieved by ultrasound disintegration in combination with trypsin and dithioerythrol treatment, but neither a GdG, peak nor a G₂+ M peak was observed in DNA histograms. A better yield of about 38% of the original nuclei number was obtained by preparation of suspensions of nuclei using citric acid and the detergent Tween 20 in combination with magnetic stirring. Both S-phase index and BrdUrd labelling index could be determined by FCM and showed the normal diurnal variations. However, the BrdUrd labelling index in suspensions of nuclei was significantly higher than the labelling index determined after immunohistochemistry. The FCM S-phase index at times of day with low DNA synthesizing activity was higher than the BrdUrd index, indicating a fraction of unlabelled S-phase cells. In conclusion, detection of incorporated BrdUrd in oral mucosa by immunohistochemical techniques or flow cytometry is feasible and provides a useful tool for fast measurements of proliferation.     

Is there an accumulation of cells during G2 in some acute lymphoblastic leukaemias?

Abstract. In some cases of acute lymphoblastic leukaemia (ALL) the percentage of cells in G₂+ M is higher than anticipated when compared with the percentage in S phase. This increase in G₂+ M, as detected by flow cytometry measurement of DNA content, may be due to an accumulation of cells, either in G ₂ or during the end of S phase; it may also be related to the existence of small tetraploid clones generally ignored by cytogeneticists. In order to identify possible subpopulations of cells with a DNA index ≥ 2-0, we have compared the results of a cytogenetic analysis to the G₂+ M values. We have also studied the distribution of S phase cells in 24 cases of ALL by incorporating 5-bromodeoxyuridine, labelling the cells by indirect immunofluorescence, and analysing them by flow cytometry after propidium iodide staining. The distribution of cells during S phase was quantified: no accumulation of cells was ever observed at the end of S phase. The question of the existence of small tetraploid clones, G₂ arrested cells or cells with a G₂ elongation remains open. However, we feel that it is more probable that, in this pathology, an elongation of the duration of G₂ occurs.     

Flow cytometric BrdUrd-pulse-chase study of heat-induced cell-cycle progression delays

The flow cytometric, bromodeoxyuridine (BrdUrd)-pulse-chase method was extended by analysing five kinetic parameters to study perturbed cell progression through the cell cycle. The method was used to analyse the cell-cycle perturbations induced by heat shock. Exponentially growing, asynchronous Chinese hamster ovary (CHO) cells were pulse labelled with BrdUrd and simultaneously heated at 43°C for 5,10 or 15 min. The cells were then incubated in a BrdUrd-free medium and, at various times thereafter, were prepared for flow cytometry. Five compartments (BrdUrd-labelled divided and undivided, and unlabelled G₁, G₁S, and G₂) were defined in the resulting dual-parameter histograms. The fraction of cells and the mean DNA content, when appropriate, were calculated for each compartment. The rates of cell-cycle progression were assessed as time-dependent changes in the fraction of cells in a given compartment and/or the relative DNA content of cells within a given compartment. Linear regression analysis of the data revealed two distinct modes of alteration in cell progression: 1 a delay in cell transit (either out of or into a given compartment), and 2 a decrease in the rate of cell transit. Hyperthermia produced a delay in the exit of cells from the G₁ compartment of ≈ 16 min per minute of heat at 43°C with no threshold. In contrast, the delay in the exit of cells from all other compartments showed a threshold of from 3 to 5 min at 43°C. Above this threshold the delay in exit of cells from the BrdUrd-labelled, undivided compartment was 25 min per minute of heat at 43°C. The more complex dose-response function of this latter compartment may reflect the fact that it includes two cell-cycle phases, S and G₂+ M. The decrease in the rate of transit out of G₂ for cells heated in G₂ was significantly larger than that for any other compartment, consistent with previous studies, which showed a G₂ accumulation following hyperthermia. These results indicate that heat exposure induces very complex alterations in cell-cycle progression and that this flow cytometric method offers a straightforward approach for observing such alterations.     

Observations regarding DNA replication sites in human cells in vivo following infusions of iododeoxyuridine and bromodeoxyuridine

In studies using bromodeoxyuridine (BrdUrd) and/or iododeoxyuridine (IdUrd) to label S phase cells in cancer patients, several unique observations were made regarding DNA replication sites and the organization of newly synthesized DNA in post-mitotic cells. While the majority of tumour specimens removed at the end of infusions demonstrated concentration of replication sites around the nuclear membrane, biopsies obtained in leukaemic patients 1 week later demonstrated several distinct patterns of labelling. For example, one, two or all lobes of granulocytes were labelled. Scavenger macrophages bearing labelled leukaemic cells in their cytoplasm were also seen. Sequential IdUrd/BrdUrd labelling of solid tumours showed various patterns of nuclear/nucleolar/membrane labelling, allowing more precise localization of early versus late replication sites.     

Turnover and Maturation Kinetics In the Hairless Mouse Epidermis. Continuous [3H]Tdr Labelling and Mathematical Model Analyses

Abstract. Hairless mice were continuously labelled with 10μCi of tritiated thymidine ([³H]TdR) every 4 h for 8 d, and the proportions of labelled basal and differentiating cells were recorded separately. the mitotic rate was measured by the stathmokinetic method and the cell cycle distributions were measured by flow cytometry of isolated basal cells at intervals during the labelling period. the mitotic rate of the [³H]TdR-injected animals did not deviate from control values during the first 5 d. Computer simulations of the data based on various mathematical models were made, and three main conclusions were obtained: (1) a large spread in transit times through the G₁ phase was found, together with a very narrow distribution in maturation time of differentiating cells; (2) about 20% of the differentiating cells were estimated to leave the basal cell layer directly after mitosis. This is consistent with results obtained from different sets of data; and (3) during continuous labelling more than 90% of the cells are labelled during each passage through the S phase.     

CELL POPULATION KINETICS OF PLUCKED AND UNPLUCKED MOUSE SKIN I. UNIRRADIATED SKIN

A detailed study of the cellular proliferation kinetics in interfollicular plucked and unplucked mouse skin has been made in Swiss albino mice, using tritiated thymidine autoradiography. Diurnal variations in mitotic and labelling indices were demonstrated in both systems.
The mean cell cycle times for unplucked and plucked skin were estimated by four different methods and found to be 100 ± 10 and 47 ± 3 hr respectively. Most of the difference was due to the shortening of G₁ phase after plucking. Repeated labelling at intervals shorter than the DNA synthesis times resulted in all the basal layer cells becoming labelled, so that the growth fraction was unity, in unplucked and plucked skin.
A well-defined second wave of labelled mitoses was seen at about 100 hr after labelling the unplucked (i.e. normal) mouse skin.
A double labelling technique using ¹⁴C-TdR and ³H-TdR with a single layer of emulsion gave reasonable values for the duration of the DNA synthesis phase.     

Influence of Growth Hormone and Thyroxine On Cell Kinetics In the Proximal Tibial Growth Plate of Snell Dwarf Mice

Abstract. In Snell dwarf mice, the influence of short-term treatment with human growth hormone (hGH) or thyroxine on the proliferative and sulphation activity of the proximal tibial growth plate was studied. By autoradiographic methods, the [³H]methylthymidine incorporation after a single injection was measured, after 2 hr incorporation time. the labelling index was calculated and the number of labelled mitoses was counted. In addition, the distribution of the labelled nuclei over the proliferating and degenerating zones was determined by continuous labelling for 25 and 73 hr.
In untreated dwarf mice after [³H]-methylthymidine administration, the number of labelled nuclei in the growth plate is low. Labelling occurs, as expected, mainly in the cells of the proliferative zones. the number of labelled nuclei in control dwarf mice was similar after 25 and 73 hr continuous labelling. This suggests that many cells are in a resting G_o or prolonged G₁ phase. Both hGH and T₄ treatment induce a significant increase of the number of labelled nuclei per growth plate and of the number of mitoses. Since hormonal treatment induces a small number of mitoses after 2 hr incorporation of the label, the minimal G₂ phase of the cell cycle is less than 2 hr. In addition, treatment with hGH and T₄ stimulates chondrocytes in the zone of proliferative and hypertrophic cells to actively incorporate [³⁵S]-sulphate.