Escape from metaphase arrest by Ehrlich ascites cells grown in vitro during treatment with 5-chloropyrimidin-2-one.
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Animals↗
Biomedical subjects
T Lindmo
Publications and source records attributed to T Lindmo.
Flow cytometry: a high-resolution instrument for everyone.
A new flow configuration for flow cytometry has been devised in which a flat, laminar stream of water, containing the stained cells in a narrow sector, is formed on a microscope cover slip by a pressurized jet of water directed onto the glass at low angle. The stream of cells is viewed by means of a fluorescence microscope with incident illumination and a pulse photometer. Coupled to a multichannel pulse height analyzer, the instrument constitutes a stable and easy-to-operate flow cytometer with a resolution equal to or better than a coefficient of variance of 1.4 percent in measurements of cellular DNA.
Animals↗
Initiation of the blastogenic response of lymphocytes by hyperoptimal concentrations of concanavalin A.
The blastogenic response of human lymphocytes in vitro to hyperoptimal concentrations of concanavalin A (Con A) has been studied by means of volume spectroscopy (measuring cellular and nuclear volume), flow cytofluorometry (measuring cellular DNA content) and incorporation of [3H]thymidine ([3H]dThd). The optimal Con A dose with respect to [3H]dThd incorporation was about 30 micrograms/ml. In cultures given hyperoptimal doses, e.g. 100 micrograms/ml, [3H]dThd incorporation was strongly inhibited, whereas the number of cells entering S-phase and significantly increasing their cellular and nuclear volume was considerably larger than with 30 micrograms/ml. With 200 micrograms/ml Con A, which induced negligible [3H]dThd incorporation, the percentage of responding cells was even larger. Hence, doses of Con A, which were hyperoptimal with regard to [3H]dThd incorporation, induced blastogenic response, including DNA synthesis, in a larger percentage of the cells than did the optimal dose. However, in cultures with hyperoptimal Con A doses, the progression of the cell cycle stagnated mainly during S- and G2-phase and few cells completed mitosis. Thus, the blocking effect of hyperoptimal doses was not confined to any particular point of the cell cycle. The reduced [3H]dTd incorporation, seen with hyperoptimal doses, is attributed partly to a failure of this assay under such conditions.
Cell Survival↗
Characteristics of a simple, high-resolution flow cytometer based o a new flow configuration.
A new flow configuration allows a flow cytometer of high resolution and stability to be assembled from an inverted fluorescence microscope with incident illumination, a pulse photometer, and a multichannel pulse height analyzer. A nozzle produces a hydrodynamically focused sample stream in a liquid jet that id directed onto a microscope cover glass in front of the microscope objective. The microscope provides a mechanically stable optical system of high numerical aperture (N.A.) (oil immersion, N.A. = 1.3) for focusing the excitation light and collection of the fluorescence light. The instrument has wide optima with regard to the various characteristics of the flow configuration, such as the rate of sample analysis and sheath flow, and the angle of incidence of the liquid jet, thus making it easy to adjust for optimal performance. DNA histograms of rat thymocytes stained with ethidium bromide and mithramycin demonstrate that all angles of incidence can be used. Large-angle incidence (70 degrees) gives the best resolution, i.e., a coefficient of variance (CV) of 0.9% of the peak of the histogram. This is only slightly better than values obtained at other angles, e.g., CV = 1.3% at vertical (0 degrees) incidence. It is concluded that instrumental resolution is equal to or better than CV = 0.9%. Linearity (proportionality between channel number and fluorescence intensity) is within 1%, and instrumental drift over a 1-h period is normally less than 1-2%.
Animals↗
Cell-cycle inhibition by misonidazole of human cells cultivated in vitro under aerobic conditions.
By means of flow cytometric recording of DNA histograms and counting of cells in synchronized populations, we have found that misonidazole (MIS) in clinically relevant concentrations induces cell-kinetic changes in human cells (NHIK 3025) cultivated in vitro under aerobic conditions. The effect seems to be a general lengthening of the cell cycle, affecting all phases. However, induction of this effect is phase-dependent, since only cells exposed to MIS during mitosis and/or early G1 will suffer significant cell-cycle prolongation. In exponentially growing populations this effect of MIS leads to a transient increase in the fraction of G1 cells and a corresponding decrease in the fraction of S cells. The possible significance of this effect for the clinical use of MIS is discussed.
Aerobiosis↗
Cell-cycle inhibitory effects of the mitotic inhibitor NY 3170 on human cells in vitro.
Effects of the mitotic inhibitor NY 3170 (1-propargyl-5-chloropyrimidin-2-one) on cell-cycle kinetics of NHIK 3025 cells were studied by means of time-lapse microcinematography, pulsed incorporation of [3H] thymidine, flow cytometry, and mitotic index. All the experiments were performed with cells synchronized by mitotic selection. Mitotic inhibition as well as inhibition in interphase was examined. The small fraction of cells able to escape mitotic arrest at 0.2mM NY 3170 had spent about 12 h in metaphase. The metaphase block was complete at 0.3 mM. For comparison, complete metaphase arrest of NHIK 3025 cells was reached at 8 mM after treatment with the parent substance NY 3000 (5-chloropyrimidin-2-one, previously reported). At 0.3mM NY 3170 interphase was also considerably prolonged. All stages of interphase were prolonged, in contrast to the interphase prolongation after treatment with high concentrations of the mitotic inhibitors vincristine and vinblastine, which occurs in G2. It was shown that the presence of NY 3170 during mitosis is a necessary and sufficient condition for metaphase arrest, thus demonstrating that metaphase arrest is not dependent on some preceding event in interphase.
Cell Cycle↗
Delay of cell cycle progression after X-irradiation of synchronized populations of human cells (NHIK 3025) in culture.
The effect of X-irradiation on the cell cycle progression of synchronized populations of the human cell line NHIK 3025 has been studied in terms of the radiation-induced delay of DNA replication and cell division. Results were obtained by flow cytometric measurement of histograms of cellular DNA content and parallel use of conventional methods for cell cycle analysis, such as pulse labelling with [3H]thymidine and counting of cell numbers. The two sets of methods were generally in good agreement, but the advantages of employing two independent techniques are pointed out. Irradiation was found to have a minor influence on DNA replication. As compared with unirradiated populations, half-completed DNA replication was 20--30 min delayed in populations 580 rad in mid-G1 or 290 rad in early S. Cell cycle progression was markedly delayed in G2. The sensitivity induction of this delay was 0.6 min/rad for populations irradiated in mid-G1, and 1.4 min/rad for populations irradiated in early S.
Cell Cycle↗
Selection of optimal model for the DNA histogram by analysis of error of estimated parameters.
The ability of four different mathematical models of the DNA histogram to give accurate estimates for the fractions of cells in G1, S, and G2 + M has been investigated. The models studied differ in the form and number of parameters of the function used to represent cells in S-phase. Results obtained from simulated DNA histograms suggest that the standard deviations of the model parameters increase exponentially with the width of the G1 and G2 + M peaks of the histogram. Error analysis is presented as a method to select a model of optimal complexity in relation to the resolution provided by the data in a given set of DNA histograms. Introduction of additional parameters improves the agreement between model and data but may result in a less well-posed model. A model with an optimal number of parameters can therefore be found that will yield parameter estimates with the smallest possible standard deviations.
Cell Cycle↗
Altered membrane-associated functions in chronic lymphocytic leukemia cells.
Peripheral blood lymphocytes consisting mainly of neoplastic B cells from patients with chronic lymphocytic leukemia (CLL cells) showed a markedly reduced response to the human B-cell mitogens anti-beta2 microglobulin, Sepharose-bound protein A and Sepharose-bound anti-human immunoglobulin (anti F(ab')2) in all of nine patients studied. On the other hand, CLL cells from three out of eight patients tested responded well to the calcium ionophore A23187. Sepharose-bound protein A and anti-beta2 microglobulin also failed to induce increased uptake of 86Rubidium (potassium analogue) in CLL cells as compared to B-cell-enriched preparations of normal peripheral blood lymphocytes. The capacity of CLL cells to cap various surface markers including beta2 microglobulin was reduced. On the other hand, surface concentrations of beta2 microglobulin were not reduced as measured by fluorescein-labelled anti-beta2-microglobulin in single-cell cytofluorometry. It is concluded that various membrane-associated events elicited by ligand-receptor interactions are altered or blocked in CLL cells.
Aged↗
The effect of colchicine and colcemid on the mitogen-induced blastogenesis of lymphocytes.
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Cells, Cultured↗
Cellular and nuclear volume during the cell cycle of NHIK 3025 cells.
The distribution of cellular and nuclear volume in synchronous populations of NHIK 3025 cells, which derive from a cervix carcinoma, have been measured by electronic sizing during the first cell cycle after mitotic selection. Cells given an X-ray dose of 580 rad in G1, were also studied. During the entire cell cycle the volume distribution of both cells and nuclei is an approximately Gaussian peak with a relative width at half maximum of about 30%. About half of this width is due to imperfect synchrony whereas the rest is associated with various time invariant factors. During S the mean volume of the cells grows exponentially whereas the nuclear volume increases faster than for exponential kinetics. Hence, although cellular and nuclear volumes are closely correlated, their ratio does not remain constant during the cell cycle. Volume growth during the first half of G1 is negligible especially for nuclei where the growth appears to be closely associated with DNA-synthesis. For unirradiated cells the growth of cellular and nuclear volume is negligible also during G2 + M. In contrast, the X-irradiated cells continue to grow during the 6 hr mitotic delay with a rate that is constant and about half of that observed in late S. Hence, radiation induced mitotic delay does not appear merely as a lengthening of an otherwise normal G2. During G1 and S the irradiated cells were identical to unirradiated ones with respect to all the parameters measured.
Cell Count↗
Effects of different growth conditions on survival after irradiation in hypoxia of human cells (NHIK 3025) in vitro.
Cell cycle kinetics and radiation response under hypoxic conditions were analyzed with human cells of the line NHIK 3025. The cells were either kept in continuous exponential growth by frequent reculturing, or went through log and plateau phase for each passage (recultured weekly). The cell cycle time for weekly recultured populations in early log phase was shorter than for cells in continous exponential growth. Cells in continuous exponential growth were more sensitive to radiation than cells in log phase. The difference in sensitivity was not due to partial synchrony of weekly recultured populations.
Cell Count↗
Inhibitory effects of the new mitotic inhibitor 5-chloropyrimidin-2-one and of vincristine on human cells in vitro.
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Cell Cycle↗
Cell cycle distributions, growth characteristics, and variation in prolactin and growth hormone production in cultured rat pituitary cells.
Clonal strains of rat pituitary tumour cells (GH3 cells) spontaneously produce and secrete prolactin and growth hormone. Chromosome analysis and DNA ploidy measurements revealed that the GH3 cells in the present study were triploid and had a decreased chromosome number compared to the parent strain. Monolayer cultures of these cells grow exponentially for 6-7 days with a mean doubling time of 54 h. Cell cycle distributions and phase durations were determined by micro-flow fluorometric measurements of cellular DNA content combined with computer calculations. During exponential growth the cell cycle distribution did not change (65.4% cells with a G1 phase DNA content, 24.9% with an S phase DNA content, and 9.7% with a (G2 + M) phase DNA content). Counting of mitoses gave 1.4% cells in M phase. The 3H-Tdr labeling indices were determined by autoradiography, and the results were in good agreement with the number of cells in S phase as calculated by micro-flow fluorometry. The phase durations were: Ts=15.9 h, TG2=6.2 h, TM=1.1 h, and TG1=30.9 h. TS and TM calculated from 3H-Tdr labeled and Colcemid treated cultures gave corresponding results. In plateau phase cultures the number of cells with a G1 DNA content increased to 80% and the number of cells with an S phase DNA content decreased to between 5% and 10%. The specific production of prolactin and growth hormone determined by radioimmunoassay showed two and four-fold increases respectively, during exponential growth. The hormone values decreased to initial or subinitial values (day 2 values) when approaching plateau phase. We conclude: that changes in the cell cycle distribution of the cell population cannot be responsible for the spontaneous alterations in hormone production during growth and that most of the hormone-producing cells must be in the G1 phase.
Animals↗
Flow cytometric measurement of the polarization of fluorescence from intracellular fluorescein in mammalian cells.
Based on the description of a laboratory-built flow cytometer, the necessary modifications of this instrument for the measurement of fluorescence polarization are described. At a maximum rate exceeding 1,000 cells/s, the instrument is capable of measuring simultaneously the horizontally and vertically polarized component of the fluorescence emitted from stained cells excited with vertically polarized light. By mathematical analysis of the accumulated data, the distribution of polarization values in the population is obtained. Various sources of instrumental error have been investigated. The large aperture of the detector optics leads to systematic underestimation of the polarization values. Other errors are negligible, and the instrument is shown to give results consistent with the theory of fluorescence polarization. Application of the instrument is illustrated by experiments with mammalian cells exposed to the fluorogenic substrate fluorescein diacetate (FDA). The polarization of the fluorescence from intracellular fluorescein produced by hydrolysis of FDA is measured, giving information on the cytoplasmic microviscosity. It appears that this microviscosity is constant over the cell cycle. On the other hand, it is significantly affected by the osmolarity of the medium.
Cell Division↗
Cell cycle characteristics of synchronized and asynchronous populations of human cells and effect of cooling of selected mitotic cells.
The method of synchronizing cells by means of mitotic selection has been adapted to the human line NHIK 3025. Increase in cell number as a function of time in asynchronous and synchronous populations was studied as well as mitotic index as a function of time after selection of synchronized populations. Phase durations of the cell cycle of synchronous populations were determined by 3H-thymidine incorporation and scintillation counting. The relative phase durations of exponentially growing asynchronous populations were determined by mathematical analysis of DNA-histograms recorded by flow cytofluorimetry. Both the generation time and the various phase durations of the cell cycle were found to be the same in asynchronous and synchronous populations. It was found that NHIK 3025 cells are damaged by cooling to 4 and 0 degrees C so that cooling of selected cells in order to increase the yield would reduce the quality of the synchronized populations.
Cell Count↗
Separation of mouse epidermal basal and differentiating cells for microflow fluorometric measurements: a methodologic study.
The DNA content of lymphocytes and of basal cells from normal hairless mouse epidermis was measured by microflow fluorometry (MFF). To obtain a relatively pure suspension of epidermal basal cells a combined mechanical and enzymatic method was used. The admixture of differentiating cells into the basal cell fraction after cell separation was 13%. The results were compared with those obtained with conventional Feulgen microspectrophotometry applied to basal cells and dermal lymphocytes in histologic sections. The results from both cytophotometric methods were in good agreement and clearly demonstrated the improved resolution obtained by using microflow fluorometry. When the lymphocytes were not treated with pepsin before being stained with ethidium bromide for MFF, the modal DNA value was consistently below that of the basal cells from the same specimen. Pepsin treatment of lymphocytes, however, increased their fluorescence intensity to the value of epidermal basal cells. The modal DNA value of Feulgen-stained dermal lymphocytes in histologic sections was consistently below that of epidermal basal cells from the same section. The advantage of pepsin treatment for obtaining higher resolution of DNA measurements of basal and differentiating epidermal cells and of lymphocytes was evaluated. The cell cycle distribution of basal cells from epidermis in different states of proliferative activity was determined. Changes in the proportion of cells in S phase were parallel to changes in the 3H-Tdr labeling index.
Animals↗
Regenerative proliferation of mouse epidermal cells following adhesive tape stripping. Micro-flow fluorometry of isolated epidermal basal cells combined with 3H-TdR incorporation and a stathmokinetic method (colcemid).
The proliferating cells of mouse epidermis (basal cells) can be separated from the non-proliferating cells (differentiating cells) Laerum, 1969) and brought into a monodisperse suspension. This makes it possible to determine the cell cycle distributions (e.g. the relative number of cells in the G1, S and (G1 + M) phases of the cell cycle) of the basal cell population by means of micro-flow fluorometry. To study the regenerative cell proliferation in epidermis in more detail, changes in cell cycle distributions were observed by means of micro-flow fluorometry during the first 48 hr following adhesive tape stripping. 3H-TdR uptake (LI and grain count distribution) and mitotic rate (colcemid method) were also observed. An initial accumulation of G2 cells was observed 2 hr after stripping, followed by a subsequent decrease to less than half the control level. This was followed by an increase of cells entering mitosis from an initial depression to a first peak between 5 and 9 hr which could be satisfactorily explained by the changes in the G2 pool. After an initial depression of the S phase parameters, three peaks with intervals of about 12 hr followed. The cells in these peaks could be followed as cohorts through the G2 phase and mitosis, indicating a partial synchrony of cell cycle passage, with a shortening of the mean generation time of basal cells from 83-3 hr to about 12 hr. The oscillations of the proportion of cells in G2 phase indicated a rapid passage through this cell cycle phase. The S phase duration was within the normal range but showed a moderate decrease and the G1 phase duration was decreased to a minimum. In rapidly proliferating epidermis there was a good correlation between change in the number of labelled cells and cells with S phase DNA content. This shows that micro-flow fluorometry is a rapid method for the study of cell kinetics in a perturbed cell system in vivo.
Animals↗