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.     

KINETICS OF NEUTROPHILIC GRANULOCYTES IN THE BLOOD OF RATS

The kinetics of neutrophilic granulocytes in the blood of rats were investigated using in vivo ³H-TdR labelling and autoradiography. The radioactive precursor was administered by single injection, repeated injections at 5 hr intervals and continuous infusion. The appearance of labelled granulocytes in the blood and in the sputum was recorded up to 120 hr after tracer application. In contrast to results after a single injection of ³H-TdR, complete labelling of the blood granulocyte pool was achieved when the DNA precursor was given by continuous infusion or four repeated injections at 5 hr intervals. In the latter experiments, an exponential replacement of unlabelled blood granulocytes by labelled granulocytes could be demonstrated, the mean intravascular half-life being 5·7 hr. This figure is in good agreement with values obtained by isotopic techniques in other mammalian species.     

DOUBLE PULSE LABELLING WITH 3H-THYMIDINE: A METHOD FOR CALCULATING CELL POPULATIO KINETICS

A method was developed for calculating cell population kinetics with the aid of double pulse labelling with ³H-TdR. The labelling index was measured from auto-radiographs of the oral epithelium of two groups of 30-day-old rats. One group was injected once with ³H-TdR the other twice, with a time interval of 90 min between injections. DNA synthesis time was calculated from the equation:
   
where T _s= synthesis time; LI₁= labelling index in the single-injected group; LI₂= labelling index in the double-injected group; and T _i= time interval between injections. Synthesis time in the palate was 4.8 hr and 7.6 hr in the tongue. Generation time was calculated from the equation: T _g= ( T _s/LI₁) × 100, where T _g= generation time. Generation time in the palate was 46.1 hr and 75.8 hr in the tongue     

INFLUENCE OF METABOLIC DNA ON DETERMINATIONS OF THE CELL CYCLE

Abstract. Evidence in favour of labelling of DNA in excess of requirements for mitosis was found in adult organs showing no mitosis (heart muscle and brain), in organs with low mitotic indexes (liver, seminal vesicle) and, more recently, in the small intestine of rodents, in bone formation and in growing roots of Vicia faba. A survey of published data showed higher labelling indexes than would be expected from the data for S, M and t _c deducted from labelled mitoses curves. to improve the accuracy of the data needed for a complete assessment the duration of mitosis (M) and the proportion of cells which are no longer in the mitotic cycle in the crypts were determined using Colcemid. the fact that all cells in the villi are derived from the crypts and that there is no cell-loss in the villi was checked by cell-counts.
The results show that 3040%of the labelled nuclei found in crypts of the jejunum of mice at 1 hr after injection of ³H-thymidine do not proceed to mitosis.
The labelling after the last mitosis is interpreted as formation of the metabolic DNA necessary for the function of the differentiated cells in the villi. There is some evidence that metabolic DNA necessary for the processes of mitosis might be lost     

CELL KINETICS IN MOUSE THYMUS STUDIED BY SIMULTANEOUS USE OF 3H-THYMIDINE AND COLCHICINE

Following an injection of ³H-thymidine to mice there is no initial incorporation in small thymocytes, only in larger ones. In the course of time small thymocytes aquire the label. Whether the delayed uptake in small thymocytes is due to a direct cell to cell transfer of labelled nuclear material from inititally labelled larger cells to small thymocytes, or whether it is due to small thymocytes being formed from larger cells by mitotic division was investigated by the administration of Colcemid® immediately after one injection of ³H-thymidine. In the absence of cell division no labelled small thymocytes appeared with time. This finding does not support the idea of a cell to cell transfer of DNA; it rather lends support to the view that small thymocytes arise by mitotic division of larger cells in the thymus. During the treatment with Colcemid® the migration of cells took place from peripheral to central cortex just like under normal conditions.     

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.     

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.     

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.     

Tracheal epithelium: cell kinetics and differentiation in normal rat tissue

Abstract. The fate of [³H]thymidine ([³H]Tdr) pulse-labelled cells was followed in tracheal epithelium of young male rats. The time course for cell differentiation, and the relation of events to tissue composition were studied. In vivo labelling and light microscope autoradiography of epoxy embedded sections were used. Labelled and total nuclei for each cell type, and combinations of labelled cells which were adjacent to one another, were tallied. Hierarchical analyses of variance were performed on the several data sets. All cell types, except ciliated, were labelled at 1 hr. A few labelled ciliated cells were seen 24 hr post-label. The frequency of labelled intermediate cells peaked at day 2; goblet and ciliated cells at day 3. No significant changes occurred in the labelling index, but at 24 hr the frequency of adjacent labelled cells (ALC) had increased > 5-fold, and changes had occurred in patterns of ALC combinations. The labelled ciliated cells which were seen at 24 hr were adjacent to labelled intermediate cells. No labelled basal-ciliated cell combinations were seen at any time. Data indicated that ciliated cells can develop from S-phase intermediate cells within 24 hr, and neither basal nor superficial goblet cells are progenitors of ciliated cells. It is proposed that both superficial goblet cell and ciliated cell development is preceded by two divisions: a basal cell division followed by an intermediate cell division.     

[3H]thymidine labels less than half of the DNA-synthesizing cells in the mouse tumour, carcinoma NT

Abstract. We have studied carcinoma NT, a transplantable mouse adenocarcinoma of spontaneous origin. Cells labelled with [³H]thymidine ([³H]TdR) were restricted to a narrow zone around the periphery of this tumour and were also found in rings up to 50 μ m wide, around isolated blood vessels in the central necrotic area. Labelling with [³H]deoxyuridine ([³H]UdR), another DNA synthesis precursor, produced a very different pattern. The labelled zone around the periphery was much wider than with [³H]TdR, and [³H]UdR labelled cells were found up to 110 μ m from isolated vessels. [³H]iododeoxyuridine ([³H]IUdR) gave the same pattern of labelling as [³H]UdR. In the heavily labelled zone, within 1 mm of the tumour periphery, the labelling index (LI) was 51% after [³H]UdR or [³H]IUdR injection, and only 36% with [³H]TdR.
The data show that at least half of the DNA-synthesizing cells in this tumour did not incorporate [³H]TdR. Previous workers reported cell loss factors for carcinoma NT of 60% calculated from [³H]TdR labelling data and 30% from the rate of loss of [¹²⁵I]UdR. The present work suggests that calculations based on [¹²⁵I]UdR data are more likely to be accurate for carcinoma NT than those using [³H]TdR data.     

Human Lymphocyte Proliferation: Dna Synthesis Time

Abstract. The DNA synthesis time ( T _s) of lymphocytes from spleens and lymph nodes of patients with Hodgkin's disease was determined by the double labeling method. ³H-TdR was administered in vivo and removed tissues minced in ¹⁴C-TdR in vitro. Lymphocytes from patients with lymphosarcoma and reticulum cell sarcoma were studied in a similar manner. Lymphocytes were divided into A cells, small with non-basophilic cytoplasm, B cells, small with basophilic cytoplasm, C cells, large with non-basophilic cytoplasm, and D cells, large with basophilic cytoplasm.
The T _s of splenic lymphocytes, in four samples not containing Reed-Sternberg cells, in hours, was: B 11.3; C , 7.9; D , 8.4; combined, 8.8. the T _s of B lymphocytes was significantly longer than that of C and D lymphocytes. A lymphocytes did not label sufficiently to measure T _s. C and D lymph node lymphocytes and lymphocytes in tissues containing Reed-Sternberg cells had a longer T _s than splenic C and D cells. the former was: B , 12.7; C , 11.7; D , 11.0; combined, 11.8. the latter was: B , 12.2; C , 11.3; D , 11.2; combined, 11.6. the T _s of Reed-Sternberg cells in one specimen of a splenic Hodgkin's tumor was 13.1 hr. Macrophage T _s was 10.7 and 15.1 hr. Lymphosarcoma cell T _s was 14.2 and 14.6 hr. Reticulum cell sarcoma cell T _s was 7.5 and 7.7 hr.
The following minimum times were calculated from observation of ³H-TdR only labeled mitotic figures: S to prophase, 71 min; S to metaphase, 75 min; S to telophase, 100 min.     

AUTORADIOGRAPHIC DETECTION OF DNA POLYMERASE CONTAINING NUCLEI IN SARCOMA 180 ASCITES CELLS

A technique has been developed allowing the autoradiographic detection of incorporation of ³H-thymidine-5'-triphosphate (³H-TTP) into nuclear DNA of smears of Sarcoma-180 (S-180) mouse ascites tumors under the direction of the cell's own nuclear DNA polymerase. Dried smears are dipped into an agar solution, which strips cytoplasm from the nuclei, and are then air dried and incubated with a buffered mixture containing four nucleotide triphosphates (one labeled), Mg⁺⁺, and Ficoll, with the cell's own DNA acting as primer. The incorporation of ³H-TTP into the nuclei, like the cell free DNA polymerase assay, is largely dependent on the presence of all four nucleotide triphosphates and Mg⁺⁺ and produces a product which is DNase sensitive and RNase resistant.
DNA polymerase activity, as studied in a cell free assay, decreases with tumor age. This correlates well with a decreasing ³H-TTP labeling index in autoradiographs of aging tumors. The ³H-TTP labeling index has also been shown to exceed but parallel the in vivo ³H-thymidine (³H-TdR) pulse labeling index for all tumor ages examined.
In at least some cell systems DNA polymerase seems characteristic of cells in cycle. The autoradiographic detection of nuclei containing the enzyme offers a new tool for the study of tumor cytokinetics.     

The proliferative response of rat liver parenchymal cells after partial hepatectomy A methodological study comparing flow cytometry of nuclear DNA content and in vivo and in vitro uptake of thymidine

Abstract. DNA synthesis in rat hepatocytes, from livers regenerating after 70% hepatectomy, was assessed by flow cytometric determination of nuclear DNA content and by incorporation of [³H]thymidine. Parenchymal liver cells were isolated by collagenase perfusion and low-speed centrifugation. Nuclei from the isolated cells were prepared for flow cytometry by a treatment with detergent, pepsin and RNase, and stained with ethidium bromide. Parallel samples of cells were incubated with [³H]thymidine and analysed for rate of incorporation of radioactivity into DNA and for labelling index determination.
The flow cytometric measure of the replicative response, i.e. the presence of cells with S-phase DNA content within the diploid and tetraploid cell populations, was compared with the incorporation of [³H]thymidine. For each of fourteen animals, including two control rats and twelve partially hepatectomized animals killed either before (at 13 hr after hepatectomy), at the onset (16 and 18 hr) or at the peak (24 hr) of regenerating activity, a fairly good correlation was found between the different methods. Satisfactory resolution of the flow cytometric detection of S-phase cells was indicated by a sorting experiment using an Ortho (system 50-H) cell sorter which demonstrated that after [³H]thymidine injection in vivo 88% of the diploid and 84% of the tetraploid S-phase nuclei were labelled, while labelling in the G¹-fractions was only 2 and 7%, respectively.     

Retention of Labelled Dna Precursor By Murine Cells After A Single Administration of Tritium Labelled Thymidine

Abstract. The central zone of the rat lens epithelium, extending half way from the centre to the periphery of a whole mount preparation, normally has less than 1% of the cells in the cell cycle at any given time. Mechanical wounding initiates a burst of proliferation in the central zone. DNA synthesis begins 14 hr after wounding followed by mitosis 10 hr later. When [³H]TdR was applied at 2 hr prior to S phase, some moderately heavy and some light labelling was observed after the onset of S phase. When [³H]TdR was applied 5 hr before S phase (9 hr after wounding), all the cells were lightly labelled. Only small amounts of the label were available to these cells 5 hr after application. It is significant that there was labelling in this group because it indicates the persistence of relatively small intracellular pools of [³H]TdR for several hours after the initial 'pulse' labelling of cells. Determinations of the duration of S phase were based on the assumption that pulse labelling may be affected by the persistence of the pools of [³H]TdR and consequent light labelling of the cells.     

The spatial organization of the hierarchical proliferative cells of the crypts of the small intestine into clusters of 'synchronized' cells

Abstract. A statistical analysis of the distribution of [³H]TdR-labelled cells in longitudinal and transverse sections of crypts from the ileum of the mouse, indicated that there was a strong tendency for labelled or unlabelled cells to be associated in short vertical runs and lateral clumps, suggesting the presence of clusters of labelled cells on the sides of the crypts. A model is discussed for the cellular spatial organization of the crypt that proposes a vertical alignment of the cells within branches of the proliferative cell lineage. The model would predict vertical alignment of partially synchronized cells as well as some lateral clumping.
In the present studies mitoses were not observed at higher levels in the crypt than labelled (S phase) cells. This observation would be predicted by the non-random spatial organization suggested by the model.
The model would also make certain predictions concerning cell migration. These are discussed in relation to cell migration studies which include evidence that migration continues in the absence of mitotic activity.     

THE DISTRIBUTION AND DIFFERENTIATION OF LYMPH-BORNE IMMUNOBLASTS AFTER INTRAVENOUS INJECTION INTO SYNGENEIC RECIPIENTS

Thoracic duct lymph from inbred, hooded rats was collected 3–5 days after antigenic stimulation of the caudal lymph nodes. During this period the lymph contained 10–15% of large, basophilic lymphoid blast cells (immunoblasts). By incubating the lymph cells at 38.C with radioactive DNA precursors, either ³H-thymidine or ¹²⁵I-deoxyuridine, the immunoblasts became labelled but the small lymphocytes did not. The lymph cells were then washed and injected intravenously into syngeneic recipients which were killed after various intervals up to 24 hr so that the radioactivity of their organs could be assayed by scintillation counting and autoradiography.
The main finding was that in animals killed after 4 or more hours the small gut always contained most of the recoverable activity and autoradiographs showed that this was because the injected cells had infiltrated the lamina propria in large numbers. Earlier, many of the injected cells were retained temporarily in the lungs, liver and spleen but many of them soon left those organs and entered the lamina propria of the small gut.
An electron microscope study of autoradiographs showed that 24 hr after injection the cells which entered the lamina propria of the gut had differentiated into plasma cells so that they displayed abundant, lamellar endoplasmic reticulum.     

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.     

THE KINETICS OF GRANULOSA CELLS IN DEVELOPING FOLLICLES IN THE MOUSE OVARY

This investigation describes the kinetics of the granulosa cells in medium-sized follicles type 3b, 4 and 5a in ovaries of 28-day-old Bagg mice. the method of labelling with ³H-thymidine followed by high resolution autoradiography is used in the experimental work, which consist of determining percentage labelled mitosis (PLM-) and continuous labelling (CL-) curves. In order to analyse the data by computer two alternative hypotheses A and B are set up. Both include the assumptions of no cell loss, exponential growth and a resting compartment Q. In hypothesis A cells from Q re-enter the mitotic cycle via the normal DNA-synthesis compartment S_p. Hypothesis B includes beside compartment S_p a special DNA-synthesis compartment S_q where only cells from Q are synthesizing DNA, and these cells re-enter the mitotic cycle via the G₂ compartment. the mean transit time in S_q is considered to be longer than the mean transit time in S_q. On the basis of the hypothesis mathematical expressions for the PLM- and CL-curves are obtained, and by means of a computer the theoretical curves are fitted to the experimental values: thereby all relevant cell kinetical parameters are estimated. Hypothesis B seems to give the best fit between the theoretical and experimental curves. the estimated parameters are: mean cycle times, μ_c= (56.1 hr, 56.1 hr and 22.3 hr for type 3b, 4 and 5a respectively), doubling times, T _D= (96.4 hr, 118.6 hr and 59.1 hr) and the proportion of cells in Q, p _Q = (0.60, 0.71 and 0.69).     

KINETICS OF THE INCORPORATION OF SOME TRITIATED NUCLEIC ACID PRECURSORS AND [3H]LYSINE INTO MOUSE BRAIN CELLS FOLLOWING INTRAPERITONEAL INJECTION

Abstract— Intraperitoneal injection into white mice of the same amount of radioactivity (0.5 mCi) of [³H]uridine and [³H]lysine demonstrated by autoradiography that there was a much greater labelling of nerve cells from lysine than from uridine. For uridine, the choroid plexus cell nuclei gave maximal labelling within 1 h, with a decrease after 6 h. The plexus nuclei of lysine-injected animals gave almost the same amount of labelling during the experimental period of 48 h. In nerve cells, labelling from uridine increased in the nuclei up to 18 h after injection and there was an almost parallel increase in the labelling in the cytoplasm and neuropil. These results are compared with earlier reports on the results from intravenous injection of uridine. In lysine-injected animals the nerve cell nuclei and cytoplasm showed a fairly constant amount of label over 48 h, but the neuropil counts increased steeply. The activity of the blood was determined by scintillation counting during the 48-h period, and, as with uridine injection, was found to be almost constant over this period. A small series of animals was injected with 0.5 mCi of [³H]uracil, [³H]guanine, [³H]guanosine or [³H]cytidine for comparison. The autoradiograms from animals injected with these bases showed very slight labelling; that from guanosine was heavy in plexus nuclei, slight in nerve cells, and from cytidine it was heavy in plexus cells and moderate in nerve cells.