KINETICS OF PHYTOHEMAGGLUTININ STIMULATED LYMPHOCYTES IN CHRONIC LYMPHOCYTIC LEUKEMIA

The DNA synthesis pattern and several kinetic parameters of in vitro PHA stimulated normal and CLL lymphocytes were determined. The DNA synthesis peak of CLL lymphocytes occurred 2–3 days later than that of normal lymphocytes. The generation time, estimated by the labeled mitoses method, was found to be 28 hr and 20 hr for CLL and normal lymphocytes respectively. This difference was mainly due to longer S and G_t periods. It was also shown that both CLL and normal lymphocytes divide several times. These data were confirmed by the chromatid labeling pattern and by the halving of the grains and the double labeling techniques. By combining continuous and pulse labeling the growth fraction of CLL lymphocytes was found to be progressively increasing, because of the recruitment of new cells in cycle, from the third day of culture. Therefore the delayed peak of DNA synthesis of CLL lymphocytes was caused by a longer cell cycle and by a longer pre-replicative phase.     

COMPARATIVE PROLIFERATIVE KINETICS OF CELLS FROM NORMAL HUMAN EPIDERMIS AND BENIGN EPIDERMAL HYPERPLASIA (PSORIASIS) IN VITRO

Proliferation kinetics of epidermal cells from normal human skin and lesions of psoriasis (benign epidermal hyperplasia) were studied in vitro. Epithelial out-growths were obtained from skin explants and the cell cycle studied using the conventional method of following two successive curves of labeled mitoses after an initial pulse with ³H thymidine. The length of T_c was 59 hr and 53.5 hr respectively for normal and psoriatic cells. The shorter T_c for psoriasis was due to a shorter duration of S. The growth fraction was 66% and 74% for normal and psoriatic cells respectively as determined by continuous labeling with ³H thymidine. Under the conditions of the present experiments, therefore, normal and psoriatic epidermal cells showed no significant difference in proliferative capacity.     

Cell Synchrony Techniques. II. Analysis of Cell Progression Data

Abstract CHO cells which have been sorted by mitotic detachment, centrifugal elutriation and fluorescence activated cell sorting have been followed for up to 14 hr by flow cytometry to examine their progression characteristics. Mathematical modelling techniques were used to provide quantitative estimates of the cell-cycle parameters. Mitotic detachment gives an 11.2-hr cycle time with mean transit times T_G1, T_s and T_G2M equal to 3.2, 5.6 and 2.4 respectively. Cells prepared by central elutriation in an early G₁ state have a 14-hr cycle time with T_G1, T_s and T_G2M of 5.7, 6.0 and 2.3 hr. Populations prepared by centrifugal elutriation enriched in early S and late S and G₂M have transit times of 2.7, 5.9 and 1.6 hr and 4.9, 6.7 and 2.1 hr with cycle times of 11.2 and 13.2 hr respectively. Cell sorting for a G₁ population gives transit times of 9.8, 8.0 and 3.6 for an overall 21.4-hr cycle time.     

EVIDENCE OF RAPID AND SLOW PROGRESSION OF CELLS THROUGH G2 PHASE IN MOUSE EPIDERMIS: A COMPARISON BETWEEN PHASE DURATIONS MEASURED BY DIFFERENT METHODS

Percentage labelled mitosis (PLM) measurements were initiated at four different times during a 24-hr period and continued for 24 hr in hairless mouse epidermis. Estimates of G₂ and S phase durations (mean T_G2 and mean T_S) were calculated. A significant number of labelled mitoses (10–20%) was seen after 30 min in all four PLM measurements and the estimated mean T_G2 varied from 1.4 to 2.5 hr and was in agreement with values from PLM measurements in other epithelial tissues. These mean T_G2 values were much shorter than expected from [³H]TdR double labelling experiments and from a multiparameter cell kinetic study in hairless mouse epidermis and did not reflect the circadian variations seen in these studies. the differences in estimates of phase durations can be explained by postulating two G₂ cell populations; one with a rapid and another with a slow rate of cell cycle progression. the cells with the higher rate are mainly registered by the PLM method, whereas those with the lower rate largely escape detection by this method. T_G2 estimates from PLM measurements in mouse epidermis therefore do not reflect the phase duration of the entire G₂ population. It is also concluded that circadian variations in T_S can not be accurately registered by the PLM method.     

Rat C-6 Glioma Cells Maintained In an Organ Culture System: A Study of Kinetic Parameters

Rat C-6 glioma cells were grown on a sponge foam matrix in an organ culture system and the cell cycle parameters, including the growth fraction (GF), were assessed after autoradiography. the zones of growth consisted of a compact upper layer (UL) at the gaseous interface, a central necrotic layer and a deeper lower layer (LL) which invaded the matrix. the fraction of continuously labeled mitoses (FCLM) was similar in both the UL and LL cells. the derivatives of the FCLM curves obtained in three experiments gave an average modal T_G2 of 5 hr. A mathematical model relating GF, T_G2, T_C and labeling index as a function of time, LI(t), was devised for cells in a steady state exposed continuously to tritiated thymidine and was applied to data obtained from UL cells. A mean GF of 9% (range: 8–10%) and a mean cell cycle time (T_C') of 27 hr (range: 13–47 hr) were obtained. the mean T_S was calculated to be 11 hr (range: 8–16 hr) by the method of grain counts per mitotic figure or grain index (GI). Knowledge of T_S permitted alternative calculation of the cell cycle time from the equation T_S/T_C= LI(0)/GF: this gave a mean cell cycle time (T_C) of 29 hr (range: 20–45 hr). Except for the GF, the cell kinetics were comparable to those of the same cell line grown in monolayer culture. the GF in the in vitro system described is in the lower range reported in some human malignant gliomas in vivo.     

ON THE EXISTENCE OF A Go-PHASE IN THE CELL CYCLE

The model is based on the assumption that the cell cycle contains a G_o-phase which cells leave randomly with a constant probability per unit time, γ. After leaving the G_o-phase, the cells enter the C-phase which ends with cell division. The C-phase and its constituent phases, the‘true’G₁-phase, the S-phase, the G₂-phase and mitosis are assumed to have constant durations of T, T₁T_s, T₂ and T_m, respectively. For renewal tissue it is assumed that the probability per unit time of being lost from the population is a constant for all cells irrespective of their position in the cycle. The labelled mitosis curve and labelling index for continuous labelling are derived in terms of γ, T, and T_s. The model generates labelled mitosis curves which damp quickly and reach a constant value of twice the initial labelling index, if the mean duration of the G_o-phase is sufficiently long. It is shown that the predicted labelled mitosis and continuous labelling curves agree reasonably well with the experimental curves for the hamster cheek pouch if T has a value of about 60 hr. Data are presented for the rat dorsal epidermis which support the assumption that there is a constant probability per unit time of a cell being released from the Go-phase.     

Non-Circadian Periodicity in the Duration of the Cell Cycle and the G1 Phase in the Hindlimb Epidermis of the Anuran Tadpole,Rana Pipiens

Using the percentage labeled mitoses method, seven cell cycle determinations were initiated at 6-hr intervals over a 36-hr span in order to see if the cell cycle in the tadpole hindlimb epidermis varied with time or showed rhythmicity. There was a pattern of two long cell cycles followed by a shorter one. Total cell cycle length (Tc) and the length of the G₁ phase plus one-half of the mitotic time (TG₁+½M) fluctuated the most, although only TG₁+½M varied significantly with the Chi-square test. The proportion of TC spent in each phase was also calculated. Only TG₁+½M/Tc had statistically significant fluctuations with time.

Rhythmicity was analyzed by a computer program using the method of least squares for cosine curve fitting. Statistically significant ultradian rhythms of 18.4 hr in TC, 18.5 hr in TG₁+½M and 18.6 hr in TG₁+½M/TC and the length of the DNA synthetic phase/total cell cycle length (TS/TC) were found. Circadian rhythmicity was not observed. The acrophases of the ultradian rhythms of TC and TG₁+½M coincided, suggesting that the rhythm of TC was due mainly to variation in TG₁+½M. In the absence of significant variation in TS, the longest phase of the cell cycle, whenever G₁+½M was short, TS/TC increased, so that the 18.6 hr rhythm in TS/TC was also a result of the periodicity in TG₁+½M.     

THE EFFECT OF ESTRADIOL ON THE CELL KINETICS IN THE UTERINE AND CERVICAL EPITHELIUM OF NEONATAL MICE

The effect of estradiol-17β on the length of the various phases of the cell cycle was studied in the neonatal mouse uterine, and cervical epithelium. A double labelling method was used, and in addition labelled mitoses were counted. In the uterus proper, estradiol shortens the length of the total cell cycle, T_c, from 17-9 hr to 15-7 hr, and the duration of S phase, T_s, from 6–7 to 5-1 hr 6 hr after estradiol treatment. 12 hr after estradiol treatment, T_c is shortened to 7-4 hr and T_s to 4–5 hr. The shortening of T_c at 12 hr is mainly due to an effect on T_G1, which is shortened from 8–55 hr in untreated animals to 1–8 hr in estradiol treated animals. The T_c of cervix epithelium cells in untreated animals was found to be 21-8 hr. After treating the mice for 6 hr with estradiol the T_c was now increased to 47 hr and further to 61 -2 hr following 12 hr treatment with the hormone. T_s increases from 8-3 hr to 15-2 hr following 6 hr estradiol treatment, and to 15-4 hr after 12 hr treatment. The effect is most pronounced in T_G1, which is lengthened from 10–95 hr in untreated animals to 28-1 hr and 43 hr, respectively, in animals treated for either 6 or 12 hr with estradiol.     

ANALYSIS OF THE GROWTH KINETICS OF A HUMAN LYMPHOMA CELL LINE

The growth kinetics of an established human lymphoma cell line were analyzed by a variety of techniques utilizing various cell inocula (5 x 10⁴ - 5 x 10⁵ cells) dispensed into 60 mm diameter dishes. Techniques included pulse-labeled mitosis (PLM), continuous labeling with ³H-TdR, time-lapse photography (TLP), cell counts by electronic particle counter, and DNA histography obtained by pulse cytophotometry (PCP). There were no significant differences among values determined for any kinetic parameters as a function of cell concentration. the average doubling time of exponentially growing cells, regardless of cell inoculum, was 44.1 hr. the generation time determined by PLM was 31.1 hr with a SD of 4.7 hr. Transit times for each stage were: T_G1= 10.6 hr, T_s= 9.9 hr, T_G2= 9.9 hr, and T_m= 0.7 hr. Repeated experiments using continuous labeling with ³H-TdR demonstrated a T_G2 of 6.3 hr. the longer value determined by PLM is possibly due to the technical manipulations of this procedure which may delay pulse-labeled cells from resuming cell cycle transit. Hence, values for cell cycle stages were recalculated to give T_G1= 14.1 hr, T_s= 9.9 hr, T_G2 = 6.3 hr, and T_m= 0.7 hr. These results were used to compute the size of each cell cycle stage compartment pool and corresponded very closely to values defined directly by PCP. TLP analysis considered only cells that produced colonies of at least thirty-two cells. Generation times ranged from 8 to 89 hr and showed a positive skewness. the average value measured for 330 divisions was 34.5 hr with a SD of 13.2 hr. Thus, the variance predicted by curve fitting of the PLM data did not correlate with that defined by time-lapse photography nor did it encompass the range in generation times observed directly by TLP. There was a positive correlation between sister-sister cell generation times (+0.66) but no relation was noted for mother-daughter values.     

THE SPERMATOGONIAL STEM CELL POPULATION IN ADULT RATS

Radioautographed whole mounted seminiferous tubules from adult rat testes were used to analyse undifferentiated type A spermatogonia at various intervals up to 81 hr following a single injection of ³H-TdR. the data obtained led to the identification of the spermatogonial stem cell and to the formulation of a new model for spermatogonial renewal and differentiation. Undifferentiated type A cells were morphologically alike, but were topographically classified as (1) isolated or (2) paired and aligned. Although labeled isolated A cells were scattered over most stages of the seminiferous epithelium, their proliferative activity varied with the stage; their labeling index was 20-30% in stages I and II, but less than 1% in stages VII and VIII. By tracing the labeled divisions of isolated A spermatogonia in time, it was seen that some daughter cells became separated from one another to form two new isolated cells, while others remained together as paired A spermatogonia. Analysis of two successive waves of labeled mitoses revealed that most paired A spermatogonia continued to proliferate forming four aligned A cells, many of which divided again to produce a chain of eight and so on. the greatest incidence of labeling among paired and aligned A spermatogonia occurred in stages XIII-III. In stage I, where the labeling index was 50%, the calculated proliferative fraction was 1 for these spermatogonia. Between stages II and V, they began to leave mitotic cycle, and during stage V this entire cohort morphologically transformed into A₁ spermatogonia. Labeled metaphase curves for undifferentiated A spermatogonia were distinct from any of the curves previously constructed for the six classes of differentiating spermatogonia, especially because of particularly long S and G₂ phases in the former. the cell cycle time of paired and aligned A cells was 55 hr, compared to an average of 42 hr for differentiating types A₂ to B.     

Comparison of in vitro and in vivo cell cycle parameters of lymphoid cells in Pig spleens

Summary Parameters of the cell cycle of lymphoid cells were estimated by analyzing percent labeled mitoses curves after a ³H-thymidine flash. Either anaesthetized pigs were labeled and multiple biopsies taken from the spleen in vivo or isolated perfused pig spleens were labeled in vitro. The data from in vivo and in vitro experiments were very similar.The mean values for cell cycle parameters were: 20.2 to 20.5 hours for the generation time, about 0.5 to 1 hour for G₂, about 1.2 to 1.3 hours for M; about 17 to 16.5 hours for S and about 1.5 to 1.7 hours for G₁. The mean grain count halving time of labeled mitoses was in accordance with the measured generation time. The isolated perfused spleen seems to give results equal to in vivo data and could, therefore, be employed as a model for studying cell cycle parameters not only in animal but also in human lymphoid tissue.The expert technical assistance of Mrs. A. Fischer is gratefully acknowledged. This study was supported by the Deutsche Forschungsgemeinschaft, SFB 112.     

A CHARACTERIZATION OF THE BOUNDARY BETWEEN THE CYCLING AND RESTING STATES IN ASCITES TUMOR CELLS

Growth deceleration of an Ehrlich ascites tumor with increasing mass is associated with a prolongation of the cell cycle and a decline in the growth fraction. These effects are reversed upon transfer of cells from an older tumor into a new host. Studies were made to locate the stages at which a cell cycle could be suspended or resumed. Transplantation caused a prompt rise in both mitotic and flash H³TdR labeling indices. When all the cells in cycle including mitoses were prelabeled with H³TdR in older tumors, the fraction of labeled mitoses did not decline for a considerable period after transplantation into new hosts. This suggests that the early rise in mitoses is not due to a flow of resting (G_o) cells from a G² store (G²-G^o transition). It appears rather to be a reflection of a lag of the mitotic process relative to other stages during the initial readjustment of the cycle. A prompt rise in flash H₃TdR indices in the transplants suggested cell entry into S from either a suspended G_I (G₁-G_o transition) or a suspended S (S-G_o transition). These possibilities were examined by relating micro-spectrophotometric estimates of DNA to the cell cycle stage as revealed by H³TdR autoradiography. Since G_o cells had DNA values corresponding to G_I, it was concluded that decycling or recycling could occur only after mitosis and before DNA synthesis.     

Cell cycle analysis and synchronization of the TN-368 insect cell line

Summary A cell cycle analysis of theTrichoplusia ni (TN-368) insect cell line is described. By means of autoradiography and percent labeled metaphase data, the cell cycle parameters were determined to be as follows: S, 4.5 hr; G₂, 8.5 hr; M, 0.5 hr; G₁, 1.0 hr; the total cell time being 14.5 hr. A synchronization procedure using 50mm thymidine in a double block procedure was used to provide a method of obtaining a large number of cells in particular cell cycle phases, especially S and G₂. This work was supported in part by U.S. Environmental Protection Agency Grant R-802516.     

Cell kinetic studies on the JB-1 ascites tumour of the mouse

Abstract. The FLM method, modified by double labelling with [³H]- and [¹⁴C]-thymidine, has been applied to the 4-day old JB-1 ascites tumour of the mouse. It results in well separated waves of purely [³H]- and purely [¹⁴C]-labelled mitoses, which show a remarkable asymmetry with long tails to the right. The following values for the mean transit times of the cells have been derived from this FLM curve, for a tumour age of 4–6 days: T_C= 32.5 hr, T_S= 16.7 hr, T_G1= 3.7 hr, T_G1= 11.0 hr and T_M= 1.1 hr. A further evaluation of the FLM curve, however, is difficult, due to the non-stationary growth of the tumour. A number of other experimental findings (growth curve, decrease of the labelling and mitotic index with increasing tumour age, two single-labelled FLM curves starting 4 and 6 days after tumour inoculation) indicate that the cell cycle time increases during the experimental period of the double-labelled FLM curve (about 2 days). A lengthening of the cycle time should result in an increasing enlargement of the areas under the waves of the modified FLM curve. However, such an increase in area has not been found; the areas are constant. All the results of the present cell kinetic studies would be consistent if it were postulated that the cell cycle time lengthens with increasing tumour age up to about 4 days after inoculation, then remains relatively constant at between 4 and 6 days and thereafter increases again. Short-term double labelling experiments suggest that this is actually the case. Under the assumption of nearly constant phase durations during the 5th and 6th day of tumour growth further conclusions can be drawn from the modified FLM curve. In particular, it follows that the transit times of the cells through successive cycle phases are uncorrelated and the variances of the transit times through a cycle phase are proportional to the duration of this phase.     

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

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

CELL KINETICS OF IRRADIATED EXPERIMENTAL TUMORS: CELL TRANSITION FROM THE NON-PROLIFERATING TO THE PROLIFERATING POOL

Parenchymal tumor cells of murine mammary carcinomas can be divided into two pools, using nucleoli as morphological ‘markers’. Cells with dense nucleoli traverse the cell cycle and divide, thus constituting the proliferating pool. Cells with trabeculate or ring-shaped nucleoli either proceed slowly through G₁ phase or are arrested in it. The role of these non-proliferating, G₁ phase-confined cells in tumor regeneration was studied in vivo after a subcurative dose of X-irradiation in two transplantable tumor lines. Tumor-bearing mice were continuously injected with methyl[³H]thymidine before and after irradiation. Finally, the labeling was discontinued, mice injected with vincristine sulfate and cells arrested in metaphase were accumulated over a 10-hr period. Two clearly delineated groups of vincristinearrested mitoses emerged in autoradiograms prepared from tumor tissue at the time of starting tumor regrowth: one group with the silver-grain counts corresponding to the background level, the other with heavily labeled mitoses. As the only source of unlabeled mitoses was unlabeled G₁ phase-confined cells persisting in the tumor, this observation indicated cell transition from the non-proliferating to the proliferating pool, which took place in the initial phase of the tumor regrowth. Unlabeled progenitors have apparently remained in G₁ phase for at least 5–12 days after irradiation.     

Growth dynamics of an amphibian tissue

By the “labeled mitoses” method of Quastler and Sherman and others, the cell cycle of the germinative zone cells of the bullfrog lens epithelium has been characterized. It has been shown that this cycle lasts approximately 83 days with the DNA synthetic phase enduring 100 hours and G₂, 11 hours. G₁ occupies over 90% of the total time. the duration of mitosis itself has not been precisely determined. the length of the synthetic phase was corroborated by double labeling with c¹⁴ and h³-thymidine. When the temperature is raised by 6°c, from 24° to 30° the cycle is compressed by 40%. When the nongerminative, central cells of bullfrog lens epithelium are activated (stimulated to undergo DNA synthesis and mitosis) by injury or through in vitro culture, the length of the cycle also appears to decrease. in the in vitro experiments the generation time, as judged by the period elapsing between two successive bursts of DNA synthesis involving the same cells, amounts to 177–190 hours at 24°c. by raising the temperature to 30°c the time from injury or isolation until the appearance of the first wave of mitosis is reduced by 20%.     

ANALYSIS OF THE PROLIFERATION KINETICS OF BURKITT'S LYMPHOMA CELLS

The in vitro proliferation kinetics of a cell line derived from a patient with American Burkitt's lymphoma were investigated at three different growth phases: lag (day 1), exponential (day 3) and plateau (day 5). The growth curve, labeling and mitotic indices, percentage labeled mitosis (PLM) curves and DNA content distributions were determined. The data obtained have been analysed by the previously developed discrete-time kinetic (DTK) model by which a time course of DNA distributions during a 10-day growth period was characterized in terms of other cell kinetic parameters. The mean cell cycle times, initially estimated from PLM curves on days 1, 3 and 5, were further analysed by the DTK model of DNA distributions and subsequently the mean cell cycle times with respect to DNA distributions during the entire growth period were determined. The doubling times were 39·6, 31·2 and 67·2 hr, respectively, at days 1, 3 and 5. The mean cell cycle time increased from 23·0 to 37·7 hr from day 3 to day 5 mainly due to an elongation of the G₁ and G₂ phases. A slight increase in the cell loss rate from 0·0077 to 0·0081 fraction/hr was accompanied by a decrease in the cell production rate from 0·0299 to 0·0184 fraction/hr. This calculated cell loss rate correlated significantly with the number of dead cells determined by trypan blue exclusion. Analysis of the number of dead cells in relation to the cell cycle stage revealed that a majority of cell death occurred in G₁ (r= 0·908; P < 0·0001). There was a good correlation between the in vitro proliferation kinetics at plateau phase of this Burkitt's lymphoma derived cell line and the in vivo proliferation kinetics of African Burkitt's lymphoma (Iversen et al., 1974), suggesting the potential utility of information obtained by in vitro kinetic studies.     

CELL KINETICS OF A CHONDROSARCOMA

The cell kinetics of the transplantable DC-II mouse chondrosarcoma have been studied by the pulse labelled mitoses method. The analysis gave the following estimates for the phases of the cell cycle: G, 10-5 hr; S, 9-5 hr; G₂, 4 hr with an intermitotic time of 23-5 hr. Consideration of the overall growth of the tumour indicated that the growth fraction and cell loss factor both had values of about 0–5. The results are compared with cell kinetic data from sarcomas and other cartilage tissues.     

STUDIES ON THE POPULATION KINETICS OF THE WALKER CARCINOMA BY AUTORADIOGRAPHY AND PULSE CYTOPHOTOMETRY

The proliferation parameters of the Walker carcinoma were estimated from both in vivo and in vitro measurements. The transplantable Walker carcinoma 256 was grown in male inbred BD1 rats. During exponential growth, 5-6 days after transplantation, a PLM curve was performed, yielding estimates of Tc ? 18.0 hr, Ts ? 6.4 hr, T_G2+M? 4.1 hr. With the double labelling technique in vitro under 2.2 atm oxygen we obtained: Tc ? 18.2hr, Ts ? 8.2 hr, T_G2+M? 2.0hr. From pulse cyto-photometry DNA content histograms the fractions of cells in the cell cycle phases were calculated using a computer program: f_G1? (47.6 ± 1.1)%, f_s? (34.1 ± 1.0)%, f_G2+M? (18.3 ± 1.5)%. These fractions remained constant between the fifth and the twelfth day after transplantation. At that time the tumour growth had already slowed down appreciably. The growth fraction determined by repetitive labelling was 0.96 on the fifth and 0.93 on the seventh and eleventh day. The cell loss factor was φ? 17% during exponential tumor growth and increased to about 100% between the tenth and twelfth day. The agreement of the cell kinetic data determined by autoradiography from solid tumours in vivo (PLM, continuous labelling) and autoradiography as well as pulse cytophotometry from in vitro experiments (excised material) was satisfactory.