Growth Fraction and Cycle Duration of Hepatocytes In the Three-Week-Old Rat

The proliferation of hepatocytes in the liver of 3-week-old rats has been investigated by autoradiographic methods. This investigation is a continuation of earlier work on the same topic (Schultze & Maurer, 1972; 1973). 21 days after birth, 102 rats received a single injection of ³H-TdR. the percentage of labelled mitoses was then determined 1 hr later and at various times throughout the interval up to 12 days after application of ³H-TdR. In agreement with earlier work, a first peak of labelled mitoses was found 7 hr after ³H-TdR injection. the area under the peak indicates an S phase duration of 8 hr. In addition a second very broad peak of labelled mitoses was found between 2 and 12 days after pulse labelling. the analysis of the results leads to the conclusion that the hepatocytes of the 3-week-old rat have a growth fraction close to 1 and a doubling time of 6–7 days. This is at variance with earlier results of Post, Huang & Hoffman (1963) and Grisham (1969) who had derived a value of 21.5 hr for the duration of the cell cycle and a value of only 0. 1–0.2 for the growth fraction of the hepatocytes.     

THE PROLIFERATION OF LYMPHOID CELLS IN GUINEA-PIG BONE MARROW

A double isotope DNA labelling method has been used to determine the duration of DNA synthesis (S) in bone marrow lymphoid cells classified by their nuclear diameters in smears. Incorporation of ³H-thymidine was confined almost entirely to marrow lymphoid cells of 8·0-15·0 μm nuclear diameter (large lymphoid cells). After exposure to ³H-thymidine in vivo and ¹⁴C-thymidine 40-104 min later in vitro , the proportion of cells labelled with ³H alone to those labelled with ¹⁴C(±³H) in radioautographic smears, plotted against time indicated the efflux from S per hour. Collectively, 28·3 ± 1·1% of all large lymphoid cells were in S and the efflux from S was 15·1% per hour. With decreasing cell size (nuclear diameter) the efflux fell progressively from 28·3% per hour (11·0 μm) to 9·2% per hour (8·0-8·9 μm) and the proportion of cells in S declined from 54·9 ± 2·3% to 14·8 ± 1·6%. Influx into S, measured in vitro by reversing the sequence of isotopes, closely resembled the corresponding efflux values in vivo relative to cell size. Most DNA synthesizing marrow large lymphoid cells belonged to a subgroup with deeply basophilic cytoplasm. The results demonstrate that basophilic large lymphoid cells in the marrow are actively proliferating and have a mean S phase duration of 6·6 hr. The largest marrow lymphoid cells (11·0 μm) proliferate most rapidly (S phase, 3·5 hr; maximum cell cycle time, 6·4 hr) while S duration is prolonged progressively to 10·9 hr for the smaller cells (8·0-8·9 μm).     

KINETICS OF GROWTH OF HAEMOPOIETIC COLONY CELLS IN AGAR

Cell kinetic parameters of mouse granulocytic and mononuclear cells growing in colonies in agar cultures have been measured. Analysis of flash and continuous labelling studies with ³H-thymidine together with determinations of colony size, growth fraction and mitotic indices, gave the following values for the phases of the cell cycle: G₁= 6·3 1·6 hr, S = 5·8 ± 1·4 hr, G₂= 1·7 ± 0·1 hr and M = 0·7 ± 0·1 hr (42 ± 8 min). No difference in the cell cycle parameters of granulocytic and mononuclear cells were found in this study.
Colonies of different size from cultures of the same age group had similar labelling indices, indicating that the size of a colony is not a function of the rate of proliferation of cells in the colony. Rather, variation in colony size is probably representative of an initial delay in the onset of colony development.     

DNA SYNTHESIS TIME IN LEUKAEMIC CELLS AS MEASURED BY THE DOUBLE LABELLING AND THE PERCENTAGE LABELLED MITOSES METHODS

Values of T _s provided by the double labelling method have been compared with those given by the percentage labelled mitoses curve for blast cells in the peripheral blood of a patient with plasma cell leukaemia and of rats bearing a transferable acute leukaemia. the double labelling method was carried out giving the first label (³H-thymidine) in vivo and the second label (¹⁴C-thymidine) in vitro with several values for the interval between the two labels. T _s was calculated by fitting regression lines to the results obtained. Data for percentage labelled mitoses were analysed by computer. For the plasma cell leukaemia values of T _s= 17.1 ± 7.0 hr and T _s= 19.8 ± 3.4 hr, and for the rat leukaemia values of 8.7 ± 1.7 hr and 9.0 ± 1.7 hr (7.1 hr corrected for exponential growth) were obtained from the percentage labelled mitoses and double labelling methods respectively. It is concluded that the double labelling method is valid for the study of cell proliferation in leukaemic blast cells.     

CYTOKINETIC ANALYSIS OF THE JB-1 ASCITES TUMOUR AT DIFFERENT STAGES OF GROWTH

The cell kinetics of the murine JB-1 ascites tumour have been investigated on days 4, 7 and 10 after transplantation of 2·5 × 10⁶ cells. The experimental data, growth curve, percentage of labelled mitoses curves, continuous labelling curves and cytophotometric determination of single-cell DNA content have been analysed by means of a mathematical model for the cell kinetics. The important result was the existence of 8% non-cycling cells with G₂ DNA content in the 10-day tumour, while only 0·2 and 0% were observed in the 7- and 4-day tumours, respectively. The doubling times determined from the growth curve were 22·8, 70 and 240 hr, respectively, in the 4-, 7- and 10-day tumours. Growth fractions of 76, 67 and 44% were calculated for the same tumour ages. The mean cell cycle time increased from 14 to 44 hr from day 4 to 7 due to a proportional increase in the mean transit time of all phases in the cell cycle. In the 10-day tumour, the mean cell cycle changed to 41 hr and T _G1 decreased to 0·5 hr. The cell production rate was 4·3%/hr in the 4-day tumour, 1·2%/hr in the 7-day tumour and 1·0%/hr in the 10-day tumour. The cell loss rates in the same tumours were 1·3, 0·2 and 0·7%/hr, respectively. The analysis made it probable that the mode of cell loss was an age-specific elimination of non-cycling cells with postmitotic DNA content.     

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.     

AUTORADIOGRAPHIC CONFIRMATION OF THE MITOTIC DIVISION OF EVERY MOUSE JEJUNAL CRYPT CELL LABELLED WITH 3H-THYMIDINE

The question was investigated of whether for crypt epithelia of the jejunum of the mouse all cells labelled after a single injection of ³H-TdR subsequently divide or whether cells exist in the crypt which synthesize metabolic DNA and, therefore, do not undergo division after labelling.
A double labelling experiment was performed with a first injection of ³H-TdR followed 1 hr later by an injection of ¹⁴C-TdR. Then from double emulsion autoradiographs of isolated squashed crypts the number of ³H-only, ¹⁴C-only and double labelled cells and mitoses were counted.
The double labelling produced a narrow, 1 hr wide sub-population of ³H-only labelled cells. This subpopulation of S cells completed its division before labelled cells were lost from the crypts by migration onto the villi. The results showed that this subpopulation of ³H-only cells completely doubled within 3 hr and then remained constant through 6 hr. From this result it was concluded that every cell labelled after a single injection of ³H-TdR divides.
From the same autoradiographs the flow rate through the end of mitosis was measured. From the flow rate and the mitotic index a mitotic duration of 0·5 hr was determined. The agreement of this measured mitotic time with the value calculated from the labelling index, mitotic index and S duration is also strong evidence that every labelled cell divides.
Both experiments show that the intestinal crypt does not contain cells synthesizing metabolic DNA.     

AUTORADIOGRAPHIC INVESTIGATION ON THE CELL KINETICS OF CRYPT EPITHELIA IN THE JEJUNUM OF THE MOUSE °CONFIRMATION OF STEADY STATE GROWTH WITH CONSTANT FREQUENCY DISTRIBUTION OF CELLS THROUGHOUT THE CYCLE

Cell kinetic parameters of cells in the crypt of the jejunum of the mouse were obtained autoradiographically. A number of different methods used in cell proliferation studies were applied to the same animal strain kept under constant conditions. In order to avoid effects of geometrical factors, squashes of isolated crypts were used.
The generation time was determined by the per cent labelled mitoses method of Quastler, modified by double labelling with ³H- and ¹⁴C-TdR. This modified method permits a more exact determination of the generation time. The duration of the cycle was 14 hr.
Double labelling experiments in which an injection of ³H-TdR was followed by an injection of ¹⁴C-TdR after 1 hr showed that the cell flux was 7.0%/hr at the beginning of the S-phase and 7.68%/hr at the end. Assuming steady state growth a constant cell flux of 7.15%/hr within the whole cycle can be derived from the measured generation time of 14 hr. These results clearly show that the crypt epithelia constitute a steady state system with constant frequency distribution of the cells throughout the cycle.
The per cent labelled mitoses method after a single injection of ³H-TdR as well as double labelling experiments with ³H- and ¹⁴C-TdR give an estimate of the S-phase of 8.0 or 7.4 hr respectively. Double determinations lead to a value of 0.54 or 0.52 hr respectively for the duration of mitosis and to values of 77% and 72%     

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.     

THE TEMPO OF LYMPHOCYTE RECIRCULATION FROM BLOOD TO LYMPH IN THE RAT

Radioactively labelled thoracic duct lymphocytes were obtained either by incubation in vitro with ³H-uridine or ¹⁴C-uridine or by giving potential donors repeated injections of ³H-thymidine finishing 17 days before thoracic duct cannulation. These labelled TDL were injected i.v. into syngeneic recipients which had been subjected to splenectomy and thoracic duct cannulation on the previous day. The tempo of lymphocyte recirculation from blood to lymph was reflected by the time at which radioactivity was recovered in the thoracic duct lymphocyte output of the recipient. This was measured by scintillation counting of 2-hourly fractional collections for 36 hr after the injection. Two lines of evidence showed that the majority of small lymphocytes which label intensely with radioactive uridine in vitro were uniform in their 'migration potential'with a modal blood to lymph transit time of 14–18 hr. By contrast the cells which were labelled in vivo with ³H-thymidine included a slower population with a modal transit time of 24–28 hr. These conclusions can be more fully interpreted in the light of recent evidence on thymic-independent ('B') lymphocytes.     

MALL LYMPHOCYTE POPULATIONS IN THE MOUSE BONE MARROW

Autoradiography and scintillation counting have been used for evaluation of lymphocyte turnover and life span in the bone marrow, peripheral blood and thoracic duct lymph of BALB/C mice. It was shown that the bone marrow contained two populations of small lymphocytes. One population was labelled 100% after 3–4 days of intensive injections of ³H-thymidine and constituted about 75% of the lymphocytes. The remaining 25% of the lymphocytes turned over at a much slower rate comparable to the rate of increase in labelled small lymphocytes of the thoracic duct. More than 10% of the small lymphocytes of the bone marrow were found to be unlabelled after 10 days of intensive injections of ³H-thymidine. Nine weeks after giving ³H-thymidine for 30 consecutive days, 8·6% of the small lymphocytes in the bone marrow remained labelled. The mean grain counts of cells in this population were comparable to those of thoracic duct lymphocytes at corresponding times. About 90% of the peripheral blood lymphocytes were found to have a slow turnover and a long life span.     

Growth pattern of the human promyelocytic leukaemia cell line HL60

Abstract. In order to characterize the growth pattern of the human promyelocytic leukaemia cell line HL60, its kinetic parameters were studied. The doubling time was calculated from serial cell counts, the duration of the various cell cycle phases from the analysis of the labelled mitoses curve, and quiescent population from continuous labelling experiments. Proliferation in culture was exponential up to a saturation density of about 3.0 × 10⁶ cells/ml, with a doubling time of 34.0 hr. The cell cycle duration was 24.3 ± 4.1 hr (SD), and that of the cell cycle phases was: G₁, 3.8 ± 2.2 hr; S, 15.1 ± 3 hr; and G₂, 5.4 ± 1.2 hr. The growth fraction was 0.85, and cell loss was restricted to the quiescent cells. The HL60 cell line, with fully characterized kinetics, provides a useful tool for the in vitro study of substances which may affect human leukaemic myelopoietic proliferation.     

Tracer dose and availability time of thymidine and bromodeoxyuridine: application of bromodeoxyuridine in cell kinetic studies

Abstract. The present experiments with [¹⁴C]-thymidine (TdR) and [³H]-bromo-deoxyuridine (BrdU) using mouse jejunal crypt cells show that the upper limit of the tracer dose of TdR is about 0.5 µg g body weight^-1 and that of BrdU is about 5·0 µg g body weight^-1. Applying these doses, the proportions of the endogenous DNA synthesis attributed to the exogenous DNA precursor are 2% and 9% respectively. For [³H]-TdR doses commonly used in cell kinetic studies this proportion is only 0-1-1.0%, a negligible quantity that does not influence the endogenous DNA synthesis. The maximum availability time of tracer doses of TdR as well as BrdU is 40 to 60 min, the majority of the precursors being incorporated after 20 min. The availability time is the same for TdR doses exceeding the tracer dose by a factor of 80, whereas it is prolonged in the case of BrdU doses exceeding the tracer dose by a factor of 50. BrdU is suitable to replace radioactively labelled TdR in short term cell kinetic studies, i.e. determination of the labelling index or of the S phase duration by double labelling. However, more studies are needed to elucidate how far BrdU can replace TdR in long term studies as shown by differences between the fraction of labelled mitoses (FLM) curves of a human renal cell carcinoma measured with BrdU and [³H]-TdR.     

Long duration of mitosis and consequences for the cell cycle concept, as seen in the isthmal cells of the mouse pyloric antrum. II. Duration of mitotic phases and cycle stages, and their relation to one another

Abstract. The kinetics of isthmal cells in mouse antrum were examined in three ways: (a) the duration of cell cycle and DNA-synthesizing (S) stage was measured by the 'fraction of labelled mitoses' method; (b) the duration of interphase and mitotic phases was determined from how frequently they occurred; and (c) mice were killed at various intervals after an intravenous injection of ³H-thymidine to time the acquisition of label by the various phases of mitosis.
The duration of the isthmal cell cycle was found to be 13.8 hr and that of the DNA-synthesizing (S) stage, 5.8 h. Estimates for the duration of the G₁ and G₂ stages were 6.8 and 1.0 hr, respectively.
From the frequency of mitotic phases, defined as indicated in the preceding article (El-Alfy & Leblond, 1987) and corrected for the probability of their occurence, it was estimated that prophase lasted 4.8 hr; metaphase, 0.2 hr; anaphase, 0.06 hr and telophase, 3.3 hr, while the interphase lasted 5.4 hr. In accordance with this, the duration of the whole mitotic process was 8.4 hr.
Ten minutes after an intravenous injection of ³H-thymidine, 38% of labelled isthmal cells were in interphase and 62% in early or mid prophase, while cells in late prophase and other mitotic phases were unlabelled. After 60 min, label was in late prophase, after 120 min, in mid telophase and after 180 min, in late telophase.
We conclude that there is overlap between some mitotic phases and cycle stages. Thus, while nuclei are at interphase during the early third of S, they are in prophase during the late two-thirds as well as during G₂. Also, nuclei are in telophase during the early half of G₁ but at interphase during the late half. Differences in nuclear diameter show that subdivision of both S and G₁ into early and late periods is practical.     

Protein Synthesis during Photocontrolled Progression of the Cell Cycle in Single-celled Protonemata of the Fern Adiantum Capillus-veneris

Protein synthesis during photoinduced, synchronous progression of the cell cycle in single-celled protonemata of the fern Adiantum capillus-veneris was studied by tracer techniques. Nuclei of the protonemata were labelled with ³H-thymidine during spore germination so that the amount of ³H incorporated into the TCA-insoluble fraction of the cells could be used as a measure of the cell number in each sample. The rate of the incorporation of ¹⁴C-amino acids into TCA-insoluble materials was not significantly varied at different stages of the cell cycle or by treatment with blue light. Extracts of cells labelled with ³⁵S-methionine at various times after the transfer from red light condition (G₀) to darkness (G₁ to S) were analyzed by two-dimensional gel electrophoresis. At least 3 of about 200 spots showed significant changes in intensity on fluorograms. Spot A (molecular weight 20,000, isoelectric point 6.3) was detectable only in early G₁, whereas spot B (molecular weight 19,500, isoelectric point 6.3) was found only in the late G₁ and S phases. When the cells were exposed to blue light before the dark incubation, the times of disappearance of spot A and appearance of spot B were advanced depending upon the progression of the cell cycle but not upon the clock time.     

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.     

KINETICS OF MURINE HAEMOPOIETIC CELL PROLIFERATION IN DIFFUSION CHAMBERS

Murine bone marrow cells were cultured in cell impermeable diffusion chambers in the abdominal cavities of mice. The kinetics of granulocyte and macrophage formation were studied by stathmokinetic and autoradiographic techniques. During the period of most rapid growth of proliferative granulocytes, their generation time and its different phases were: t _c∽ 8 hr, t _G1∽ 1·5 hr, t _s∽ 5·5 hr, t _G2∽ 0·7 hr and t _M∽ 0·25 hr.
The generation time of macrophages and their precursors was approximately 8 hr. Formation of macrophages was significantly reduced when chamber inoculum was increased, as judged by ³H-TdR labelling index.     

CELL PROLIFERATION IN THE CASTRATE MOUSE SEMINAL VESICLE IN RESPONSE TO TESTOSTERONE PROPIONATE

The cell proliferation kinetics following induced DNA synthesis in the mouse seminal vesicle were measured after treatment with testosterone propionate. Fraction labelled mitosis curves at 24, 48 and 72 hr after injection gave t ₂ values of 1·5, 2·0 and 1·8 hr respectively, and t _s values of 10·5, 8·0 and 8·0 hr. T _c measured 48 hr after stimulation was 17·5 hr. Growth fraction rose from 0·14 at 24 hr to 0·64 at 48 hr, and fell to 0·32 by 72 hr. A simple model is proposed in which the rise and fall of mitotic index and labelled index is determined by the 'cell distribution ratio'.     

Effect of Vinblastine On the Cell Cycle and Migration of Ameloblasts of Mouse Incisors As Shown By Autoradiography Using 3H-Thymidine

Abstract. The effects of vinblastine on the cell cycle and the migration of ameloblasts were studied in the lower incisors of mice by labelling the cells with ³H-thymidine ([³H]TdR) and radioautography. A group of mice received 2 μg/g of body weight vinblastine intraperitoneally and 6 hr after these animals and those of a control group were injected with 1μCi/g body weight of [³H]TdR, and sacrificed at time intervals from 0.75 hr to 15 days.
The generation time of ameloblasts in the progenitor compartment was 14.8 hr in animals treated with vinblastine and 17 hr in the controls, using the FLM curve method; with the grain dilution method the duration was respectively 29.25 hr and 25.96 hr. the thymidine labelling index of the treated animals was 50% higher than the controls. the velocity of ameloblast migration, determined either by the displacement of the most incisally labelled cell or by the grain dilution method, was lower in the experimental group (2.48 cell positions/hr and 9.18 μ/hr respectively) as compared with the control (3.21 cell positions/hr and 18.88 μm/hr respectively).
The results on the ameloblast production rate are contradictory but the slowing down in the velocity of cell migration is compatible with a decrease of the rate of cell production in the progenitor compartment as a vinblastine effect.     

Vegetalization of Sea Urchin Larvae Induced with Cycloheximide

The embryos, kept at 20°C for 3 hr–6 hr from the time of fertilization (at the morula stage), were cultured in sea water containing cycloheximide (10–16 mM) for successive 3 hr and then transferred to normal sea water. The embryos, thus treated, became vegetalized larvae. With the same treatment performed at a developmental stage prior to 3 hr of fertilization, most of embryos developed to small blastulae filled with mesenchyme-like cells. The treatment at a stage after 6 hr of fertilization yielded normal plutei. From the embryos exposed to both ¹⁴C-leucine and ³H-thymidine during the treatment, labelled chromatin was isolated. Only in the presumptive vegetalized embryos obtained by the cycloheximide treatment of morulae, ratio of ¹⁴C-radioactivity found in proteins of chromatin to ³H-radioactivity in DNA was markedly lower than that observed in chromatin from control embryos. The rate of ³H-radioactivity-decrease by DNase I treatment was higher in chromatin isolated from the presumptive regetalized embryos than that observed in chromatin isolated from control ones. Probable failure of chromatin structure formation, due to cycloheximide-inhibition of chromatin protein synthesis, seems to disturb the determination in the embryos at the morula stage, resulting in an induction of vegetalized embryos.