Cell kinetic analysis of a murine macrophage cell line

For the present study, which was performed to find a reliable method suitable for determination of the cell kinetic parameters of a continuous cell line, use was made of the macrophage cell line J774.1. The doubling time of the cell population was approximately 27 h. The continuous labeling curve showed that all the cells divide and almost no quiescent cells occur. The cell-cycle time as determined from the curve of the labeled cells in mitosis, the course of the stathmokinetic index, and time-lapse videorecordings, was about 19 h. The discrepancy between the population doubling time and the cell-cycle time must be due to death and disintegration of cells during culture in vitro. The results indicate that the doubling time of a cell population is not a reliable parameter to determine the kinetics of a population of continuously proliferating cells and that determination of the course of the stathmokinetic index offers a rapid and simple method to establish the cell-cycle time reliably.     

A reappraisal of the stathmokinetic technique. II. Metaphase degeneration and its correction

The rate at which proliferating cells enter mitosis is an important cytokinetic measure. It is estimated by the metaphase arrest (or stathmokinetic) technique. This technique depends on the persistence of arrested metaphases in the tissue in question. But arrested metaphases do not, in fact, persist for long; some estimates of the rate at which cells are entering mitosis, based on a count of metaphases, are therefore underestimates. This paper presents a mathematical analysis of this phenomenon and a method of calculating the initial metaphase index from the depleted index at any given time.     

Nonparametric analysis of stathmokinesis

The nonparametric analysis of the stathmokinetic experiment presented in this paper is an extension of procedures by Jagers and Staudte. The method allows one to estimate, under very general assumptions, the first two moments of the residence time in successive cell cycle phases. Approximate formulae for the mean square errors of the estimates are derived. Applications include experimental stathmokinetic data for various cell lines, both analyzed and not analyzed previously. Comparison proves that the nonparametric method is very accurate whenever it can be applied. Results of analysis of the stathmokinetic data are also discussed from the viewpoint of the variability of the cell cycle generation time.     

Iron deficiency depresses cell proliferation in hamster cheek pouch epithelium

Iron deficiency anaemia was induced in hamsters by feeding a low iron diet coupled with weekly bleeding. To assess cell proliferation, the stathmokinetic agent vinblastine sulphate was administered and cell birth rates were calculated from cumulative mitotic indices. The rate was significantly reduced in epithelium from iron-deficient animals. The uptake of tritiated thymidine [( 3H]TdR) was also significantly reduced in these animals. Results of both stathmokinetic and labelling experiments indicate that cell production in the cheek pouch epithelium of iron-deficient animals is impaired.     

Some effects of bleomycin on the proliferation, maturation time and protein synthesis of hairless mouse epidermis.

Hairless mice were given 2 mg Bleomycin i.p. in 1-0 ml saline on two successive days. By a stathmokinetic method, by micro-flow fluorometry and by autoradiography certain kinetic parameters were measured during 10 days after the last injection. Cell counts were made and the turnover time of the differentiating cells estimated. Protein synthesis was estimated by the uptake of radioactive histidine, and dry cell mass measured by weighing. Bleomycin affected cell proliferation in the epidermis by depressing biphasically both the number of cells in, and the passage of cells through, the cell cycle phases: S, G2 and M, most probably by directly affecting late G1 cells and cells in mitosis. The time between the two minima of depressed DNA synthesis corresponded to the mean generation time of the basal cells. Histidine uptake and dry cell mass were slightly affected, but the turnover time of the differentiating cells was prolonged. Bleomycin thus had a strong long-lasting inhibitory effect on epidermal cell proliferation and a marked inhibitory effect on epidermal cell maturation in mice.     

Cell cycle time and rate of entry of cells into mitosis in the small intestine of young rats

Cell cycle time (T(C)) and the rate of entry of cells into mitosis (r(M)) in the jejunum and duodenum of young rats were investigated using the stathmokinetic method. The cell cycle times in the jejunum were 24.3 and 28.3 h in light and dark periods, respectively. Cell cycle times in the duodenum were 17.1 and 21.5 h in light and dark periods, respectively. Rates of entry of cells into mitosis in the jejunum were 1.2 and 1.1 cells/cell/h in light and dark periods and rates of entry of cells into mitosis in the duodenum were 1.4 and 1.8 cells/cell/h in light and dark periods, respectively. Although these changes to cell cycle time values are not statistically significant, the variation between the two periods should be considered in relation to its possible biological effects.     

Iron deficiency depresses cell proliferation in hamster cheek pouch epithelium

Abstract. Iron deficiency anaemia was induced in hamsters by feeding a low iron diet coupled with weekly bleeding. To assess cell proliferation, the stathmokinetic agent vinblastine sulphate was administered and cell birth rates were calculated from cumulative mitotic indices. The rate was significantly reduced in epithelium from iron-deficient animals. The uptake of tritiated thymidine ([³H]TdR) was also significantly reduced in these animals. Results of both stathmokinetic and labelling experiments indicate that cell production in the cheek pouch epithelium of iron-deficient animals is impaired.     

SOME EFFECTS OF BLEOMYCIN ON THE PROLIFERATION, MATURATION TIME and PROTEIN SYNTHESIS OF HAIRLESS MOUSE EPIDERMIS

Hairless mice were given 2 mg Bleomycin i.p. in 1-0 ml saline on two successive days. By a stathmokinetic method, by micro-flow fluorometry and by autoradiography certain kinetic parameters were measured during 10 days after the last injection. Cell counts were made and the turnover time of the differentiating cells estimated. Protein synthesis was estimated by the uptake of radioactive histidine, and dry cell mass measured by weighing. Bleomycin affected cell proliferation in the epidermis by depressing biphasically both the number of cells in, and the passage of cells through, the cell cycle phases: S, G₂ and M, most probably by directly affecting late Gj cells and cells in mitosis. The time between the two minima of depressed DNA synthesis corresponded to the mean generation time of the basal cells. Histidine uptake and dry cell mass were slightly affected, but the turnover time of the differentiating cells was prolonged. Bleomycin thus had a strong long-lasting inhibitory effect on epidermal cell proliferation and a marked inhibitory effect on epidermal cell maturation in mice.     

Epidermal chalones and squamous cell carcinomas. The growth inhibitory effects of aqueous epidermal extracts (G1 and G2 chalones) on the epidermis and on a transplantable keratinizing carcinoman in nude mice.

Balb/c/nu nude mice that had been transplanted with a moderately differentiated squamous cell carcinoma were injected i.p. with different doses of epidermal chalone, and control animals were injected with saline. The labelling indices (H3TdR) and the mitotic rate (stathmokinetic method with vinblastine sulphate) were determined. In the untreated animals, both the labelling index and the mitotic rate of the tumor were considerably higher than in the epidermis, and the rate of cell birth was almost twice that of the epidermis. Higher doses of chalone were needed to reduce the labelling index for the tumour than for the epidermis, and there was generally a less pronounced dose/response relationship in the tumours than in the epidermis. The same was true of the mitotic rate but here the results were not as obvious as for the labelling index. A possible explanation of the results may be that the tumour cells are less sensitive than epidermal cells to the injected chalones, or that reduced vascularization of the transplanted tumour may lead to reduced access of chalone, or that tumour necrosis may pay a role. However, it is evident that the tumour cells react less than the epidermis to both the G1 and the G2 chalone, and thus the findings of this study do not provide any evidence against the theory that epidermoid transplanted tumours are less sensitive to epidermal chalones than normal tissue of the same histogenetic origin.     

Cell population kinetic profile of the mouse thymus, and the changes induced by prednisolone.

The cell population kinetic parameters of the thymus in BALB/c mice have been estimated using stathmokinetic and [3H]TdR techniques in both control animals and animals treated with prednisolone. FLM data were analysed by computer using the Gilbert program. The study showed that prednisolone had an inhibitory effect mainly in the DNA synthesis phase and in G1. Stathmokinetic data also showed a decrease in the cell birth rate and an increase in the apparent cell cycle time (or potential doubling time) after treatment. The labelling index, the mitotic index and the growth fraction were also decreased. The study also shows a good agreement between the data obtained by stathmokinetic and [3H]TdR techniques.     

Circadian variation in the mitotic rate of the rat corneal epithelium. Cell divisions and migration are analyzed by a mathematical model

A stathmokinetic method was used to study the diurnal variation in the mitotic rate (MR) of the rat corneal epithelium, and in the adjacent conjunctival epithelium. A prominent circadian variation in cell proliferation was observed in both epithelia, both showing almost the same pattern, which may indicate that both tissues are submitted to the same regulatory mechanisms. The average rate of cell renewal during a 24 h period indicated a mean cell renewal time of 12.3 days. This is longer than previously assumed. The MR declined toward the central cornea. Based on the above observations and the known centripetal migration of cells in the corneal epithelium, we have developed a mathematical model showing isomorphism with the renewal of the corneal epithelium.     

CELL POPULATION KINETIC PROFILE OF THE MOUSE THYMUS, AND THE CHANGES INDUCED BY PREDNISOLONE

The cell population kinetic parameters of the thymus in BALB/c mice have been estimated using stathmokinetic and [³H]TdR techniques in both control animals and animals treated with prednisolone. FLM data were analysed by computer using the Gilbert program. The study showed that prednisolone had an inhibitory effect mainly in the DNA synthesis phase and in G₁. Stathmokinetic data also showed a decrease in the cell birth rate and an increase in the apparent cell cycle time (or potential doubling time) after treatment. The labelling index, the mitotic index and the growth fraction were also decreased. The study also shows a good agreement between the data obtained by stathmokinetic and [³H]TdR techniques.     

Kinetic analysis of drug-induced G2 block in vitro

The data on cell-cycle effects of two prospective antitumour agents, (+)-1,2,-bis(3,5-dioxopiperazine-1-yl)propane (soluble ICRF; NSC 169780) and 1,4-bis(2'chloroethyl)-1,4-diazabicyclo [2.2.1] heptane diperchlorate (CBH; NSC 57198) were used to determine whether a modified stathmokinetic experiment could predict the effects of continuous, long-term (0-48 hr) drug exposure in an in vitro L1210 murine leukaemia cell system. Generally, continuous drug exposure of exponentially growing cells does not provide sufficient quantitative information concerning cell-cycle-phase-specific mechanisms of drug action. Alternatively, stathmokinetic experiments, which are usually limited to some fraction of one cell doubling time, provide little information about long-term drug effects. By using mathematical models constructed for this purpose, however, stathmokinetic data can predict the overall proportion of cells affected by a drug though failing to discern between various kinds of drug action (e.g. reversible v. irreversible block, blocking v. killing action, etc.), especially when it occurs in G2 phase. In addition, it can be shown that for at least one of the drugs (soluble ICRF) the stathmokinetic experiment fails to predict 'after-effects' of drug treatment which extend into the following cell cycle(s). It also becomes clear that the degradation of exponential growth characteristics of quickly dividing cells during long-term, continuous drug exposure makes prediction of cell-cycle kinetic perturbations uncertain when derived from short-duration stathmokinetic experiments. However, with care, the joint application of 'short term' (e.g. stathmokinesis) and 'long term' (e.g. continuous exposure) techniques allow adequate quantitative insight into drug-perturbed cell-cycle kinetics. The applicability of modelling techniques is discussed: in the present instance it is limited to lower drug concentrations. For higher drug concentrations, effects like increased ploidy, ineffective division, etc., make it impossible in the present study to obtain a clear picture of the kinetics.     

CELL-PRODUCTION RATES ESTIMATED BY THE USE OF VINCRISTINE SULPHATE AND FLOW CYTOMETRY I. AN IN VITRO STUDY USING MURINE TUMOUR CELL LINES

A method for the evaluation of cell-production rates is described which combines flow cytometry (FCM) and the stathmokinetic method. By means of FCM it is possible to estimate the distribution of cells with G₁, S and (G₂+ M) DNA content in a population. As this method gives the relative (G₂+ M) DNA content of cells within the cell cycle, it may be possible to evaluate cell-production rates by this technique. In the present study it was found that administration of a metaphase-arresting (stathmokinetic) agent, vincristine sulphate (VS), to asynchronous cell populations of three different murine tumour cell lines in vitro increased the peak representing cells with (G₂+ M) DNA content as the number of mitotic (M) cells increased during the period of treatment. The accumulation of mitotic cells was determined by cell counts on smears under the microscope and compared with the increase in the (G₂+ M) DNA peak measured by FCM as a function of time after the administration of VS. Good agreement was obtained between the cell-production rates as estimated by FCM and by mitotic counts in all three cell lines investigated.     

Human colorectal tumours in short-term organ culture A stathmokinetic study

Abstract. Short-term organ culture, using a technique to preserve epithelial/stromal interaction and metabolism, is a useful technique for carrying out kinetic studies on human colorectal carcinoma and adjacent normal mucosa, providing initial perturbations of proliferative indices are allowed to settle. Tumours require 3.0 μg/ml vincristine for complete metaphase arrest compared with mucosa, which needs 0.5 μg/ml, a 6-fold difference. Using a stathmokinetic technique, the birth rate of tumour cells is 10.21 cells/1000 cells per hr, compared with 7.73 cells/1000 cells per hr for mucosa, a statistically significant difference ( P < 0.01).     

CELL POPULATION KINETICS IN THE RAT JEJUNAL CRYPT

Cell kinetics in the jejunal crypt of the male Wistar rat were studied using autoradiographic techniques with tritiated thymidine and a stathmokinetic technique with vincristine. The migration rate measured by following the movement of the 50 % peak on the labelling index distribution curve with time after injection of tritiated thymidine gave a value of 1.43 ± 0.14 (SE) cell positions per hour, compared with a value from a cumulative birth rate of 1.78 cell positions per hour. The crypt column length was 32.9 ± 0.2 cells and the column count was 22.3 ± 0.2. This measurement gave a total crypt population of 734 cells, compared with an estimate of 650 ± 6 from direct observation of squashed, microdissected crypts. In each crypt 22.5 ± 0.5 mitoses were present, and the crypt cell production rate was 32 cells per crypt per hour; this latter value was confirmed using two independent techniques. The crypt growth fraction calculated from the durations of phases of the cell cycle and the labelling index was 0.62. A value of 0.61 was found from the labelling index distribution curve. As assessed from crypt squashes, there were 403 proliferating cells per crypt.     

Mathematical models of hierarchically structured cell populations under equilibrium with application to the epidermis

Abstract. There are three categories of keratinocytes in the germinative compartment of the epidermis – stem, transit-amplifying and post-mitotic. Their population structure is hierarchical. This means that stem cells differentiate into transit-amplifying cells which, after a few rounds of division, become post-mitotic cells. The cell processes of birth, differentiation, death and migration affect the composition and proliferation rate of the germinative compartment. These phenomena are quantified by various cell kinetic parameters. In this paper we derive equations that relate these parameters for different models of hierarchically structured cell populations in equilibrium. We include in the models asymmetric and symmetric division, variations in cell-cycle times, apoptosis and variation in the number of transit generations. We conclude that variation in cell-cycle times need only be considered if apoptosis is not negligible. If it is negligible, then only average cell-cycle times are needed. Unfortunately, it is impossible to predict the importance of apoptosis from the available experimental data. However, the strength of its effect is determined by the other parameters, especially the fraction of cycling stem cells. We show that variation in the number of transit generations can have a potentially large effect on cell birth rate. We also show that cell birth rate does not directly depend on the mean transit-amplifying cell-cycle time, only on the mean stem cell-cycle time. We argue that 'homogeneous cell population' equations should not be used to study hierarchical cell populations as has been done in the past. Finally we argue that stem cell parameters and transit-amplifying cell parameters should not be lumped together.     

B lymphocyte production in the bone marrow of mice with X-linked immunodeficiency (xid)

CBA/N mice carry an X-linked recessive immunodeficiency (xid) gene manifested by the absence of a B lymphocyte subpopulation, but the manner in which the xid gene exerts its effect on B lymphocyte development is unknown. The production of B lymphocytes in the bone marrow of CBA/N mice has now been compared with that of normal CBA/J mice by using two in vivo assays: immunofluorescence stathmokinetic studies measured pre-B cell proliferation, whereas radioautographic [3H]thymidine labeling was used to evaluate small lymphocyte turnover. Although the total cellularity of CBA/N mouse bone marrow was greater than normal, the absolute number of marrow small lymphocytes, pre-B cells, and B lymphocytes were all similar to those in CBA/J controls. Furthermore, in the bone marrow of CBA/N mice, the proliferation rate of pre-B cells, calculated from their rate of entry into mitosis, and the turnover rate of small lymphocytes, derived from their rate of [3H]thymidine labeling, were not significantly different from those seen in nondefective mice. The present findings that pre-B cell proliferation and small lymphocyte production proceed at similar rates in the bone marrow of xid and normal mice suggest that the xid gene does not act at the level of primary B cell genesis in the bone marrow. The findings are in accord with the view that the xid gene produces a maturation block or a functional abnormality among B lymphocytes in the peripheral lymphoid tissues rather than the deletion of a sublineage of B lymphocytes in the bone marrow.     

Kinetic Analysis of Drug-Induced G2 Block In Vitro

The data on cell-cycle effects of two prospective antitumour agents, (+)-1,2,-bis(3,5-dioxopiperazine-l-yl)propane (soluble ICRF; NSC 169780) and 1,4-bis(2′chloroethyl)-1,4-diazabicyclo [2.2.1] heptane diperchlorate (CBH; NSC 57198) were used to determine whether a modified stathmokinetic experiment could predict the effects of continuous, long-term (0–48 hr) drug exposure in an in vitro L1210 murine leukaemia cell system. Generally, continuous drug exposure of exponentially growing cells does not provide sufficient quantitative information concerning cell-cycle-phase-specific mechanisms of drug action. Alternatively, stathmokinetic experiments, which are usually limited to some fraction of one cell doubling time, provide little information about long-term drug effects. By using mathematical models constructed for this purpose, however, stathmokinetic data can predict the overall proportion of cells affected by a drug though failing to discern between various kinds of drug action (e.g. reversible v. irreversible block, blocking v. killing action, etc.), especially when it occurs in G₂ phase. In addition, it can be shown that for at least one of the drugs (soluble ICRF) the stathmokinetic experiment fails to predict ‘after-effects’ of drug treatment which extend into the following cell cycle(s). It also becomes clear that the degradation of exponential growth characteristics of quickly dividing cells during long-term, continuous drug exposure makes prediction of cell-cycle kinetic perturbations uncertain when derived from short-duration stathmokinetic experiments. However, with care, the joint application of ‘short term’ (e.g. stathmokinesis) and ‘long term’ (e.g. continuous exposure) techniques allow adequate quantitative insight into drug-perturbed cell-cycle kinetics. the applicability of modelling techniques is discussed: in the present instance it is limited to lower drug concentrations. For higher drug concentrations, effects like increased ploidy, ineffective division, etc., make it impossible in the present study to obtain a clear picture of the kinetics.     

Turnover and maturation kinetics in the hairless mouse epidermis. Continuous [3H]TdR labelling and mathematical model analyses

Hairless mice were continuously labelled with 10 microCi 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.