PERTURBED CELL PROLIFERATION KINETICS OF THE EMT6/M/AC TUMOUR IN MICE TREATED WITH ANTI-MOUSE LYMPHOCYTE SERUM

The cell proliferation kinetics of the EMT6/M/AC tumour were determined at two volumes, 6-3 mm³ and 180 mm³, in mice treated with anti-mouse lymphocyte serum, AMLS. Comparison of the growth curve with that obtained in non-AMLS treated animals showed a marked increase in the growth rate at all volumes in the treated group. In contrast, the cell cycle time and the intermitotic phase times were not significantly different in the treated and untreated groups at comparable volumes. The increase in the growth rate in AMLS treated mice was obtained in spite of decreases in both the rate constant for cell production and the growth fraction, and was due to a marked decrease in the rate constant for cell loss.     

CHANGES IN CELL PROLIFERATION KINETICS OCCURRING DURING THE LIFE HISTORY OF MONOLAYER CULTURES OF A MOUSE TUMOUR CELL LINE

Following inoculation of monolayer cultures of EMT6 mouse tumour cells at 10⁵ cells, a short lag is followed by 3 days of exponential growth with a population doubling time of 12 hr. A plateau cell number is reached between days 4 and 5 and is maintained for at least 8 days. During exponential growth, the pulse ³H-TdR labelling index is 55–60%, all cells are in cycle, and the median cycle time is 11–12 hr. For the first 3 days of plateau phase, the labelling index is about 25 % and there is considerable cell loss. The cell cycle is 32–40 hr, and S-phase is very long. Later in plateau phase, the labelling index falls to <2 % and there is little cell loss. The changes in kinetics occurring in EMT6 cultures are discussed with reference to reported changes occurring in other cell lines.     

KINETICS OF CELL PROLIFERATION IN THE PRE-IMPLANTED MOUSE EMBRYO IN VIVO AND IN VITRO

The cell proliferation of pre-implanted mouse embryos was investigated after development in vivo and in vitro. The studies were started at the pronuclear stage, 2 h post conception (p.c.) and continued until the hatching of blastocysts, 120–144 h p.c. The number of cell nuclei, the DNA content of each nucleus, the mitotic index and the labelling index were determined. From these data it was possible to calculate the length of the cell generation cycle and its various phases. With the exception of the first cell cycle the S-phase was constant. The G₁- as well as the G₂-phase varied in length during the different cell cycles. From 31–72 h p.c. the increase in cell number was exponential. After cultivation in vitro this increase was smaller than in vivo. At later periods the proliferation rate decreased with proceeding development. In late blastocysts most of the cells were in the G₁-phase. The development of the embryos was somewhat faster in vivo than in vitro. But in principle conditions were comparable.     

PROLIFERATION KINETICS OF AN EXPERIMENTAL ASCITES TUMOUR OF THE MOUSE

The cell cycles of an experimental ascitic tumour of the C3H mouse (NCTC 2472) were determined at various times after the intraperitoneal injection of 10⁶ cells. It was found that, contrary to results in solid NCTC 2472 tumours, obtained with the same NCTC cells, the duration of the cell cycle and its phases lengthened with the age of the tumour while the growth fraction remained relatively constant. G₁ was the first phase to lengthen, while later T_s and T_G2 increased also. The amount of DNA per cell was determined by cytospectrophotometry. This method provides data on the evolution during growth of the relative number of cells in each phase of the cell cycle.     

CELL KINETICS IN THE SEBACEOUS GLANDS OF THE MOUSE. II. THE GLANDS DURING HAIR GROWTH

The sebaceous glands of the mouse have been studied during hair growth initiated either spontaneously or artificially. The labelling index of the glands increases early in the spontaneous hair growth period. That of the epidermis is much lower and hardly changes during the growth period. After the initiation of hair growth by plucking, changes in cell proliferation in the sebaceous glands appear to follow those in the epidermis. The size of the gland and the number of cells in it also change after plucking. These variations can be related to the stages of hair growth.     

泽蛙(Rana limnocharis)活体内骨髓细胞增殖动力学的研究

离体培养的动物细胞增殖规律已有不少的报道,但是两栖类活体内细胞增殖过程如何,尚未见报道。本文运用SCD术,对泽蛙活体内骨髓细胞增殖动力学进行了较系统地观察,最后推算出Tc为12—24小时。     

KINETICS OF TISSUE PROLIFERATION IN COLORECTAL MUCOSA DURING POST-NATAL GROWTH

The main developmental event in the colorectal mucosa during post-natal growth is a dramatic increase in the number of crypts of Lieberkühn, resulting from a longitudinal fission of pre-existing crypts. In the present study, the kinetic aspects of this process have been analysed, using extensive gland and cell counts involving the entire colon and rectum of 24 male BD IX rats distributed into four age groups. The number of crypts was found to rise from an average 4652 to 423,800 between birth and adulthood; the corresponding ratios of bifurcating glands were 13.55 and 0.67%, respectively. Crypt production attained its maximum 18 days after birth with an hourly increment of 519 units. the time spent by replicating glands in the bifurcating stage (‘fission time’) averaged 6.9–10.5 hr. The mean number of epithelial cells per crypt rose from 249 in 4-day old rats to 635 in adults. the estimated total number of epithelial cells in the colon and rectum was one million in newborns and 248 million in adults. the increment in cell number peaked 3 weeks after birth with a value of 310,000/hr. During the first few days after birth, all cells produced in the epithelium were retained. Cell loss thereafter rapidly progressed, reaching 70% of the cell production in 3-week old animals.     

FIBROBLAST CELL KINETICS IN THE PERIODONTAL LIGAMENT OF THE MOUSE

Twelve male mice were injected intraperitoneally with tritiated thymidine. Six were sacrificed after 1 hr and six after 7 days. The right manibular incisors were dissected, cut sagittally and dipped in liquid emulsion. In the exposed and stained slides, observation was restricted to the lingual side of the periodontal ligament. Cells were evaluated sagittally from the basal tooth end up to the distance of 5 mm and up to the depth of 100 μm in the direction of socket wall. Cell and grain count was evaluated separately in 100 × 10 μm rectangles, creating a two-dimensional array onto which the periodontal ligament was mapped. The progenitor compartment extends up to the distance of 2400 μm from origin. Fibroblasts leaving this compartment migrate at different velocities, creating a velocity profile across the ligament. Adjacent to the socket wall cell movement is sluggish, whereas the fastest cell movement is exhibited by cells located 20–30 μm from the tooth. The existence of such a profile indicates a continuous renewal of intercellular bonds, consistent with a process of actively pulling the incisor from its socket by the migrating fibrocytes.     

CELL KINETICS OF GROWTH CARTILAGE IN THE RAT TIBIA II. MEASUREMENTS DURING AGEING

A number of cell kinetic techniques using labelled thymidine and autoradiography have been applied to study growth cartilage in the rat tibia during ageing. No change in the duration of the synthesis phase was found from 4 to 13 weeks of age but there was a reduction in cell proliferation rate during this period. Measurements of labelling index, proliferation zone size and height of hypertrophic cells were used to calculate the growth rate of the bone from 7 days to 1 year. The results agreed well with radiographic measurements of bone growth.     

ANALYSIS OF THE KINETICS OF GRANULOSA CELL POPULATIONS IN THE MOUSE OVARY

The kinetics of granulosa cell populations in two types of follicles in ovaries of 28-day-old Bagg mice are investigated. The analysis includes estimations of mean values and standard deviations of the transit times (T_G1, T_S, T_G2 and T_C), the doubling time T_D, and the proliferative fraction p. First the percentage of labelled mitosis curve (PLM-curve) and the continuous labelling curve (CL-curve) are estimated. Then a hypothesis concerning the cell kinetics of the granulosa cells in the two follicle types is set up. The normal distribution is chosen to simulate the probability density functions of the transit times. On the basis of the hypothesis mathematical expressions for the PLM- and CL-curves are worked out. By fitting the calculated PLM-curve to the experimental one it is possible to estimate mean values and standard deviations of T_G1, T_S>, and T_G2. As a test of the hypothesis the CL-curve is calculated by means of the estimated parameter values and compared to the experimental one. The calculated PLM- and CL-curves are found to be in good agreement with the experimental data as far as both follicle types are concerned. It is concluded that the method is a useful procedure. The choice of a normal distribution does not imply a significant limitation of the method in these investigations. Moreover it is concluded that the hypothesis is plausible. This means, e.g., that the proliferative fraction is unity in the two follicle types and that there is no cell loss from the cell systems.     

THE STATHMOKINETIC METHOD IN VIVO TIME-RESPONSE WITH SPECIAL REFERENCE TO CIRCADIAN VARIATIONS IN EPIDERMAL CELL PROLIFERATION IN THE HAIRLESS MOUSE

Groups of hairless mice were injected i.p. with a stathmokinetic dose of 0·15 mg colcemid at seven different times of the day and animals killed 0, 15 and 30 min, 1, 2, 3 and 4 hr after the injection. The proportion of cells in metaphase and ana/telophase was determined in histological sections. The results showed a transient accumulation of metaphases about 30 min after the injection, followed by an increase in metaphases from 1 to 4 hr. Therefore, no value before 1 hr after the colcemid injection should be used in calculations of the mitotic rate. The presence of circadian rhythms with high mitotic activity in the morning and low activity in the evening was confirmed. It is shown by regression analyses that the accumulation period of 4 hr is sufficiently short to reflect circadian variations in epidermal cell proliferation and that the 4-hr accumulation value alone is sufficient to estimate the mitotic rate.     

KINETICS OF CELL RENEWAL, CELL MIGRATION AND CELL LOSS IN THE HAIRLESS MOUSE DORSAL EPIDERMIS

Forty hairless mice were given injections of tritiated thymidine every 4th hour during 10 days. At 24 hr intervals groups of four mice were killed. The numbers of labelled basal and differentiating cells were determined by autoradiography with a stripping film technique. To determine the background activity skin sections from uninjected control mice were subjected to the same stripping film procedure. Another group of hairless mice was given one single pulse labelling with tritiated thymidine. The number of labelled mitoses was scored for 12 hr after the injection. At 10, 12 and 15 hr after the injection, the numbers of labelled basal and differentiating cells were also determined. A mathematical model of cell population kinetics in the epidermis has been suggested. The results of different simulations on this model were compared with the observed results. The curve of mean grain counts under continuous labelling increased from day to day with two well-defined plateaux. The percentage of all labelled cells increased rapidly up to the 3rd day, and thereafter the curves gradually flattened off. When basal cells and differentiated cells were considered separately the labelling index of the basal cells increased rapidly for the first 3 days and then flattened off at the 100% level on the 5th day. The labelling index of the differentiating cells was low during the first 3–4 days. Then a steep increase in the percentage of labelled differentiating cells was seen, but the curve flattened off again close to the 100 % level after the 7th day. The labelled mitosis curve had its maximum 5 hr after the thymidine injection. The curve fell again to almost zero at 12 hr. Ten, 12 and 15 hr after the injection, 6, 7 and 7% respectively of the labelled cells were found in the spinous layer. It was concluded that three grains over each nucleus could be used as lower limit for considering a cell as labelled. On this basis, tritiated thymidine injections every 4th hour can be considered as continuous labelling.     

IN VITRO PROLIFERATION OF MOUSE LYMPHOBLASTOID CELL LINES: GROWTH MODULATION BY VARIOUS POPULATIONS OF ADHERENT CELLS

The in vitro growth pattern of a number of mouse lymphoblastoid tumour cell lines was modified in the presence of adherent cell layers from various sources. The AVRij-1 and ST-4b cell lines exhibited a concentration—dependent growth pattern, i.e., they would only grow well when seeded at high starting cell concentrations. Better growth of these cells from low cell concentrations was observed in the presence of adherent cell layers from syngeneic or allogeneic bone marrow. Adherent cell layers derived from mouse spleen and pleural or peritoneal cavity could also promote the growth of the above tumour cells, but in a narrower range of cell concentrations and to a lower extent. Moreover, confluent adherent layers from the pleural and peritoneal cavities completely inhibited the growth of AVRij-1 and ST-4b cells, while adherent cell layers from the bone marrow did not inhibit growth at any cell concentration tested. The in vitro growth of concentration—independent cell lines was also affected by the presence of adherent cells from the bone marrow. Under syngeneic conditions, a slight increase in the growth of the ‘null’ or pre-B lymphoma cell line ABLS-8.1 was observed. On the other hand, the growth of tumour cells expressing more differentiated properties, such as the thymus T lymphoma tumour cell line ST-1.3 and the plasma cell tumour MPC-11.45.6.2.4, was inhibited in the presence of syngeneic bone marrow derived adherent cell layers. This inhibition was more pronounced under allogeneic conditions. Growth inhibition was also observed when concentration—independent cell lines were co-cultured with adherent cells from the pleural and peritoneal cavities. Thus, adherent cell layers from non-haemopoietic sources inhibited the growth of all cell lines tested. On the other hand, adherent cells from the bone marrow had a differential effect on growth of lymphoblastoid tumour cell lines. This depended on the in vitro growth properties of each tumour cell line and on some additional specific tumour cell properties. The latter could relate to the differentiation stage characterizing each tumour cell line. The culture method described here may serve as a model system for studies on interaction of leukaemic cell and the haemopoietic microenvironment.     

INDUCTION OF CELL PROLIFERATION IN THE MOUSE BLADDER BY 4-ETHYLSULPHONYL-NAPHTHALENE-1 -SULPHONAMIDE

The reaction of the bladder epithelium of mice, following stimulation with a carcinogen, 4-ethylsulphonylnaphthalene-1-sulphonamide (ENS), was studied. A wave of cell division was induced in the resting epithelium, diploid and tetraploid cells being the main dividing elements; some of the high ploidy surface cells also underwent division. The cell cycle time for the diploid and tetraploid cells appeared to be identical. There was considerable variation in the intensity and timing of the onset of cell division in the bladder epithelium of individual animals. ENS caused severe damage to the surface cells of the bladder epithelium as indicated by increased lysosomes and the formation of large vacuoles.     

THE EFFECTS OF RED BLOOD CELL EXTRACTS ON THE PROLIFERATION OF ERYTHROCYTE PRECURSOR CELLS, IN VIVO

Saline incubation extracts of mature erythrocytes were assayed in vivo by a variety of techniques in order to study their ability to modify the proliferation of maturing erythroid cells. Using comparable extracts from granulocytes and lymphocytes, the specificity of the effect of the red cell extract for erythroid cells was confirmed by measurement of autoradiographic labelling indices, radio-iron incorporation and spleen colony growth. The erythroid cells were found to be very sensitive to the effects of the extract, as little as 10 μg per mouse producing a maximum effect on iron incorporation. It was found that the extract does not block erythroid cell proliferation completely but simply lengthens the cell cycle, mainly by increasing the G₁ phase of the cycle. There was no effect on the committed erythroid precursor cells. The in vivo activity, specificity and non-toxicity to the cells, together with the cells' sensitivity to red cell extract suggest, therefore, that this inhibitor may play a physiological role in the control of red cell production.     

CELL KINETICS IN THE SEBACEOUS GLANDS OF THE MOUSE. I. THE GLANDS IN RESTING SKIN

Cell proliferation, differentiation and migration have been studied in the sebaceous glands of DBA-2 mice in the resting (telogen) phase of hair growth. Cells labelled by a single injection of tritiated thymidine start to leave the glands of adult male mice 5 days later. About 80% of the proliferative cells in the basal layer have a cell cycle time of 40 hr or less. In 18% of the proliferative cells G₁ is at least 4 days long and 16% have a G₂ phase longer than 17 hr. The S phase is about 7.5 hr long and cells spend at least 21 hr in the basal layer before migrating into the differentiating cell region. The glands of mature female and immature mice are smaller than those of the mature male. They have fewer, smaller cells and a much lower labelling index.     

THE DISTINCT STEPS OF CELL DETACHMENT DURING DEVELOPMENT OF MOUSE UROEPITHELIAL CELLS IN THE BLADDER

In developing mouse urinary bladder the urothelial cells respond to urine accumulation by cell detachment, i.e. desquamation. To elucidate the steps of urothelial cell detachment during embryonic development and first urine accumulation in bladder lumen, superficial and intermediate cell layers were investigated. Different electron microscopic and cytochemical methods have been used. It was possible to distinguish between an early and late events on the basis of morphology. At the beginning cell detachment involves interruption of tight junctions between neighbouring superficial cells and the formation of numerous endocytotic vesicles. Endocytotic and cup-shaped vesicles then fuse and form large parallel rows of vacuoles and multivesicular bodies in desquamating superficial cells and in the neighbouring superficial and intermediate cells. These observations clearly demonstrate the existence of specific steps during the detachment of embryonic uroepithelial cells. Apoptosis accompanies cell desquamation. Only predetermined superficial bridge cells die in an apoptotic manner.     

THE KINETICS OF CELL PROLIFERATION IN THE TRACHEOBRONCHIAL EPITHELIA OF RATS WITH AND WITHOUT CHRONIC RESPIRATORY DISEASE

Labelling indices of the tracheobronchial epithelia of conventionally-derived rats with chronic respiratory disease (CRD) and minimal-disease rats without CRD have been determined. The duration of the DNA synthesis phase (t_s) computed from the percentage of mitoses labelled at various intervals of time after injection of tritiated thymidine was 7 hr: t_G2 was 3.5 hr. Using the measured value of t_s and the labelling indices, the mean turnover times of the tracheobronchial epithelia in three groups of six 5-week-old conventionally-derived rats were calculated to be 11.2, 14.6 and 22.4 days, while in similar groups of 5-week-old minimal-disease rats the turnover times were found to be 24.3, 36.5 and 41.6 days. The majority of cell divisions in the tracheobronchial epithelium of these minimal disease rats were probably required for growth rather than renewal. The mean turnover time of this tissue in 5-week-old Syrian hamsters was 73 days. The cells of the rat tracheobronchial epithelium have been classed as basal or superficial, depending on their shape and proximity to the basement membrane. The mean turnover time of the basal cells in 5-week-old minimal-disease rats was 11.7 days calculated from labelling indices. The migration method of Brown & Oliver (1968) gave a similar value for the basal cells in minimal-disease rats, and a value of 9.5 days for the basal cells in a group of conventionally-derived rats. The mean turnover time in the latter was only 5.4 days if two rats with tracheobronchitis were included. Consideration of the slow rate of fall in mean grain count over labelled cells at intervals of time after labelling and the calculated turnover times suggests that the proliferative fraction of the basal cell population is close to unity. Well-labelled cells were still present in both basal and superficial populations in the minimal-disease rats at 10 days after labelling. The marked effects of CRD on cell proliferation in this epithelium are emphasized and the significance of this in relation to published work is discussed.     

ERYTHROPOIETIC CELL POPULATION CHANGES DURING THE HEPATIC PHASE OF ERYTHROPOIESIS IN THE FOETAL MOUSE

Estimates have been made of the absolute numbers of hepatogenic erythropoietic cells from 12.5 days post fertilization onwards in the mouse. All stages of maturation up to reticulocytes are present in the earliest samples but the least mature cells (proerythroblasts and basophilic erythroblasts) predominate; more mature cells (orthochromatic erythroblasts, reticulocytes and erythrocytes) predominate later in development. The number of hepatogenic haemoglobinized cells increases exponentially with a population doubling time of about 8 hr until about 15.5 days post fertilization. There is then a sharp transition and the doubling time lengthens to about 2 days. The immature cells formed during the rapid phase of increase are poorly haemoglobinized; hence the increase in haemoglobin lags behind that of cells. Calculations of the rates of formation of hepatogenic haemoglobinized cells and haemoglobin per standard number of liver cells show maxima between 15 and 16 days; these findings are in accord with direct observations of rates of haemoglobin synthesis in cultured mouse foetal livers made previously.     

A COMPARISON OF IN VIVO AND IN VITRO STATHMOKINETIC METHODS FOR THE STUDY OF CELL PROLIFERATION IN HAMSTER ORAL EPITHELIA

Mitotic indices (MI) expressed as numbers of metaphase figures per 100 basal cells in the cheek pouch and palatal epithelium of the Syrian hamster following metaphase arrest with vinblastine sulphate (VLB) were compared using in vivo and in vitro techniques. The MI in vivo 4 1/2 hr after intraperitoneal injection of 4 mg VLB/kg body weight was 2·69 ± 0·37 for cheek pouch and 12·08 ± 1·09 for palate. MI in vitro was measured using small tissue explants cultured for 4 hr in medium supplemented with VLB at concentrations ranging from 6-600 μg/ml. The maximum MI for cheek pouch epithelium in vitro (2·7) did not differ significantly from that observed in vivo (P > 0·50) and was obtained in the presence of 12–30 μg VLB/ml, a concentration comparable with that used in vivo. In contrast, the maximum MI for palate epithelium in culture (5·6) was significantly lower than that in vivo (P < 0·001) and was only achieved in the presence of extremely high concentrations of VLB. Possible reasons are discussed for the discrepancy between the MI for palatal epithelium in vivo and in vitro.