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.     

The role of protein accumulation in the cell cycle control of human NHIK 3025 cells

The cell cycle kinetics of NHIK 3025 cells, synchronized by mitotic selection, was studied in the presence of cycloheximide at concentrations (0.125-1.25 μM) which inhibited protein synthesis partially and slowed down the rate of cell cycle traverse. The median cell cycle duration was equal to the protein doubling time in both the control cells and in the cycloheximide-treated cultures at all drug concentrations. This conclusion was valid whether protein synthesis was continuously depressed by cycloheximide throughout the entire cell cycle, or temporarily inhibited during shorter periods at various stages of the cell cycle. These results may indicate that cell division does not take place before the cell has reached a critical size, or has completed a protein accumulation-dependent sequence of events. When present throughout the cell cycle, cycloheximide increased the median G1 duration proportionally to the total cell cycle prolongation. However, the entry of cells into S, once initiated, proceeded at an almost unaffected rate even at cycloheximide concentrations which reduced the rate of protein synthesis 50%. The onset of DNA synthesis seemed to take place in the cycloheximide-treated cells at a time when the protein content was lower than in the control cells. This might suggest that DNA synthesis in NHIK 3025 cells is not initiated at a critical cell mass.     

The relation between protein accumulation and cell cycle traverse of human NHIK 3025 cells in unbalanced growth

Human NHIK 3025 cells, synchronized by mitotic selection, were given 2 mM thymidine, which inhibited DNA synthesis without reducing the rate of protein accumulation. After removal of the thymidine the cells proceeded towards mitosis and cell division, with an S duration 2 hours shorter than, but a G2 and M duration nearly identical to that of the control cells. If cycloheximide (1.25 m?M) was present together with thymidine, no net protein accumulation took place during the treatment, and the subsequent duration of S, G2, and M was similar to that of the untreated cells. The shortening of S seen after treatment with thymidine alone would therefore indicate that the rate of DNA synthesis depended on the amount of some preaccumulated protein. The postreplicative period in thymidine-treated cells was lengthened by cycloheximide treatment although the protein content had already been doubled. This suggests that proteins required for the traverse of this part of the cell cycle might have to be synthesized after completion of DNA replication. Shortly after removal of thymidine, the rate of protein accumulation declined markedly, indicating the existence of some mechanism for negative control of cell mass. In addition, the daughters of thymidine-treated cells had their cell cycle shortened by 2 hours. As a result, the cells had returned to balanced growth already in the first cell cycle following the induction of unbalanced growth. In conclusion, our experiments suggest that NHIK 3025 cells might require a minimum time in order to traverse the cell cycle, which is independent of cell mass.     

Photodynamic effects of Photofrin II on cell division in human NHIK 3025 cells

Human cervix carcinoma cells of the line NHIK 3025 were exposed to light after 18 h incubation with Photofrin II. After this photodynamic treatment cells in the interphase were retarded with respect to entry into mitosis for a period which increased with increasing light dose. Following the prolonged interphase, an increase in the mitotic index was observed, giving rise to a 3-fold higher level of mitotic cells compared to the control level. Staining of methanol-fixed cells with the DNA-specific dye mithramycin indicated that the increase in mitotic index was due to a prolongation of the metaphase. For all the light doses studied most of the metaphase cells could be characterized as three-group metaphases or c-metaphase-like structures for the first 8 h after treatment. An approximately 10-fold increase above the control level in the number of tripolar mitoses was also observed. A 2h incubation in a Photofrin II-free medium after the 18 h incubation with Photofrin II and before light exposure reduced the fluorescence of the cells by 30 per cent. However, this wash-out period had no effect on the increase in mitotic index after light exposure. A light dose corresponding to 80 per cent survival (as assayed on asynchronous cells) was given to cells in mitosis after Photofrin II incubation. This treatment delayed more than 90 per cent of the metaphase cells from entering the anaphase for at least 1 h. Cells photodynamically treated in the anaphase and telophase entered the interphase at a similar rate as control cells. These observations indicate a temporary block in the initiation of the anaphase and a prolongation of the metaphase. A microscopic study of cells immunologically stained for beta-tubulin 1 h after photodynamic treatment indicated that the organization of the spindle apparatus was disturbed by the photodynamic treatment. Such perturbations are suggested to be the cause of the observed accumulation of cells in mitosis.     

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 traverse and protein metabolism in human NHIK 3025 cells: the role of anchorage

We have studied the effect of cell anchorage on the human cell line NHIK 3025 in vitro, to see whether the growth regulating effect of cell anchorage primarily affected DNA division cycle or mass growth cycle. It was found that cell to cell anchorage had the same effect on cell cycle progression as anchorage to a solid surface, which indicates that it is anchorage per se and not cell shape that is important for growth control in NHIK 3025 cells. When NHIK 3025 cells were grown without attachment to a solid surface, both G1 and cell cycle duration was prolonged by 6 h, which means that the prolonged cell cycle was due to a prolonged G1. During the first part of the cell cycle the rate of protein synthesis and degradation was constant, and at the same level in cells grown with and without attachment. This means that the prolonged G1 was not due to a reduced protein accumulation or mass growth. Towards the end of the cell cycle protein accumulation was reduced. This effect was either due to a size control before cell division or a secondary effect of the prolonged G1. We therefore conclude that cell anchorage as a growth regulator primarily affects the DNA/cell division cycle.     

Effect of two pyrimidine analogs on accumulation of tubulin in NHIK 3025 cells

Summary Accumulation of tubulin as compared with the accumulation of total cellular protein in human NHIK 3025 cells treated with the sulfone 2-(2-thenyl)sulfonyl-5-bromopyrimidine (NY 4137) and the sulfoxide 2-(2thenyl)sulfinyl-5-bromopyrimidine (NY 4138), two mitotic inhibitors, were investigated by two-parametric flow cytometry. Following a 4 h treatment with NY 4137 tubulin accumulation is inhibited while total protein continues to accumulate. After treatment for 4h with NY 4138 the accumulation of total protein is approximately constant, while the accumulation of tubulin is reduced although not to the same degree as that found for NY 4137-treated cells. In addition, the percentage tubulin SH-groups (6.89 ± 0.14) remaining after treatment of purified rat brain tubulin with NY 4137 or NY 4138 was determined. Treatment with 0.0125 mM NY 4137 reduced the number of tubulin SH-groups detectable with dithiobis benzoate or from 6.89 ± 0.14 before treatment to about 4 after treatment. However, practically all SH-groups of tubulin remain detectable following treatment with the same concentration of NY 4138. From the results described in this report we infer that NY 4137 binds to tubulin SH-groups and that inhibition of tubulin accumulation follows as a secondary effect.     

Cell cycle-specific glucocorticoid growth regulation of a human cell line (NHIK 3025)

It has been reported that the human cell line NHIK 3025 has a specific cytoplasmic glucocorticoid receptor. When these cells were exposed to glucocorticoids, the cell cycle time was prolonged. Cells, synchronized by mitotic selection, were subjected to the synthetic glucocorticoid dexamethasone throughout the cell cycle. Only cells exposed in the first half of G1 phase had a lengthened cell cycle time. Most of the prolongation was also located within the G1 phase. The dexamethasone growth inhibition was reversible and could be detected only in the cell cycle where the cells were exposed to the steroid. DNA-histograms of asynchronous cells were recorded by flowcytometry at various times after steroid exposure. These histograms also showed G1 phase sensitivity and G1 phase prolongation after exposure to dexamethasone. Our results thus indicate that these cells have a dexamethasone-sensitive restriction point in mid-G1 phase of the cell cycle.     

Protein synthesis as a function of protein content in exponentially growing NHIK 3025 cells studied by flow cytometry and cell sorting

Methods of cell preparation and quantitative flow cytometric cell sorting were developed for precise measurements of the incorporated radioactivity in cells labelled with [¹⁴C]-valine and fractionated as a function of their fluorescence intensity after staining with fluorescein-iso-thiocyanate (FITC). FITC fluorescence intensity of exponentially growing NHIK 3025 cells was found to be directly proportional to cellular protein content as measured by saturation-labelling with [¹⁴C]-valine. Protein synthesis as measured by ¹⁴C incorporation after pulse-labelling was found to increase nearly proportionally with cellular protein content. The deviations from proportionality were not greater than 6%, but yet found to be significant since the measurement error corresponded to only 2% standard deviation.     

Progress through G1 and S in relation to net protein accumulation in human NHIK 3025 cells

We have investigated whether human NHIK 3025 cells are dependent upon a net increase in cellular protein content in order to traverse G1 and S. The increase in DNA and protein content was studied by means of two-parameter flow cytometry using populations of cells synchronized by mitotic selection. By adding 1 μM cycloheximide to the medium protein synthesis was partially inhibited, resulting in negligible net accumulation of protein. The cells were able to enter S and progress through S under such conditions. The latter was the case whether the cells had been accumulating protein during G1 or not. The results further indicate that the larger cells enter S earlier and traverse S at a higher rate than the smaller cells. Our conclusion is that net accumulation of protein does not seem to be a prerequisite for traverse through G1 and S, i.e. DNA replication may be dissociated from the general growth of cell mass.     

Oxygen- and temperature-dependent cytotoxic and radiosensitizing effects of cis-dichlorodiammineplatinum(II) on human NHIK 3025 cells in vitro

The radiosensitizing effect of the chemotherapeutic drug cis-dichlorodiammineplatinum(II) (cis-DDP) was tested on human NHIK 3025 cells cultivated in vitro. cis-DDP was found to exert a radiomodifying effect under hypoxic but not under aerobic conditions. These results confirm that cis-DDP may act as a radiosensitizer of hypoxic cells; however, the radiosensitizing effect was seen only at concentrations of cis-DDP having a considerable cytotoxic activity, and for practical reasons concerning survival level the highest drug concentration that was investigated was 15 microM at 37 degrees C. The radiosensitizing effect was of a dose-modifying type and with a dose-modifying factor (DMF) of 1.2 at 15 microM in hypoxic cells. The radiosensitizing as well as the cytotoxic effect of cis-DDP was found to be strongly temperature dependent. Isoeffect doses of cis-DDP was reduced with a factor of 3 at 22 as compared to 37 degrees C. We also found that hypoxic cells were less sensitive to cis-DDP than cells treated in the presence of oxygen. To test the correlation between cytotoxicity and radiosensitization on the one hand and cellular uptake of cis-DDP on the other, cell-associated Pt was measured by atomic absorption spectroscopy. From these studies the cytotoxicity of cis-DDP at 22 and 37 degrees C under aerobic conditions was found to be the same as long as the amount of cell-associated Pt (i.e., the cellular uptake) was the same. However, whether the cells were treated under hypoxic or aerobic conditions, the cellular uptake of Pt was the same. While the radiosensitizing effect was present at 37 and at 40 degrees C, no such effect could be found at 22 degrees C. Since the cytotoxicity of cis-DDP as well as the drug uptake was reduced about three times at 22 as compared to 37 degrees C, we increased the concentration threefold, to 50 microM at 22 degrees C. Still no radiosensitizing effect was found at this temperature.     

Estimation of circadian variations in cell cycle phase durations in murine epidermal basal cells

Circadian variations in the proliferative activity of squamous epithelia are well known. However, circadian variations in the duration of the various cell cycle phases (S, G2 and mitosis) have been disputed. The percent labelled mitoses method, which is traditionally used to obtain duration of cell cycle phases, is poorly suited for identification of circadian variations. Therefore methods combining changes in compartment size (cell cycle phase) and cellular flux through the compartments have been used. Three different methods using such data are presented. These incorporate various simplifying assumptions that cause methodological errors. Limits for use of the different methods are indicated. The use of all three methods gives comparable and pronounced circadian variations in the duration of S and G2 phase. These results are also compatible with circadian variations in the mitotic duration, but they may also represent artefacts due to sensitivity to model errors.     

A change in the oxygen effect throughout the cell-cycle of human cells of the line NHIK 3025 cultivated in vitro.

NHIK 3025 cells were synchronized by repeated mitotic selection. The S-phase was determined by 3H-thymidine incorporation and scintillation counting. By comparing the age-response surves of aerobic cells irradiated with 500 rad with those of extremely hypoxic (less than4 p.p.m. O2) cells irradiatedwith 1500 rad, it was found that the sensitizing effect of oxygen was not constant throuhgout the cycle. It was significantly higher in S, G2 and mitosis than in G1. No significant sensitizing effect of 120 p.p.m. O2 (compared with less than4 p.p.m.O2) was found on cells in G1 when the cells were irradiated with 1500 rad. In S, G2 and mitosis, however, the sensitizing effect of oxygen at 120 p.p.m. is considered to be significant. Experiments performed with cells irradiated with 2000 rad incontact with either less than4 p.p.m. O2 or 80 p.p.m. O2 showed the same trend, little sensitizing effect in G1 and higher in S, G2 andmitosis. Dose-response curves for cells in mid-G1 and mid-S under aerobic and extremely hypoxic conditions were well fitted by the formula S=exp (-alphaD-betaD2). From the dose-response curves it was conculded that the change in the sensitizing effect of oxygen throughout the cell-cycle only appeared for low doses (in the dose region where alpha dominates). The sensitizing effect of oxygen on cells in mid-G1 was found to be increasing with increasing dose.     

Growth of cell populations with arbitrarily distributed cycle durations. II. extended model for correlated cycle durations of mother and daughter cells

A model is proposed that describes the growth of cell populations, in which the cycle durations of mother and daughter and of sister cells can be correlated. The model accounts for arbitrary frequency distributions of cycle durations and for arbitrary correlations. Depending on the mother-daughter correlations, the frequency distribution of cycle durations either remains the same or changes from one cell generation to the next one. Both phenomena are described in the literature for different cell populations. Sister-sister correlations are shown to influence only numerical values in the model but not the model's structure. Model calculations with different types of correlations are compared with growth data on the ciliate Tetrahymena geleii.     

The cytoplasmic origin of variability in the timing of S phase in mammalian cells.

The time at which S phase begins in mammalian cells is highly variable with respect to cell age. Evidence is presented that this variability does not arise because the initiation of DNA synthesis depends on the stochastic interaction of an initiator substance with a rare initiation site. Instead, the signal responsible for starting S phase must appear at random in the cytoplasm and may be transient.     

Quantitative analysis of cell cycle phase durations and PC12 differentiation using fluorescent biosensors

Cell cycle analysis typically relies on fixed time-point measurements of cells in particular phases of the cell cycle. The cell cycle, however, is a dynamic process whose subtle shifts are lost by fixed time-point methods. Live-cell fluorescent biosensors and time-lapse microscopy allows the collection of temporal information about real time cell cycle progression and arrest. Using two genetically-encoded biosensors, we measured the precision of the G1, S, G2, and M cell cycle phase durations in different cell types and identified a bimodal G1 phase duration in a fibroblast cell line that is not present in the other cell types. Using a cell line model for neuronal differentiation, we demonstrated that NGF-induced neurite extension occurs independently of NGF-induced cell cycle G1 phase arrest. Thus, we have begun to use cell cycle fluorescent biosensors to examine the proliferation of cell populations at the resolution of individual cells and neuronal differentiation as a dynamic process of parallel cell cycle arrest and neurite outgrowth.     

DNA-synthesizing cells in oral epithelium have a range of cell cycle durations: evidence from double-labelling studies using tritiated thymidine and bromodeoxyuridine

Abstract Mouse tongue epithelium is characterized by a circadian variation in the number of DNA-synthesizing cells (labelling index, LI). Cells undergoing DNA synthesis were labelled with tritiated thymidine ([³H]TdR) at 0300 (peak LI) or 1200 h (low LI). The fate of these cells was assessed by injecting animals with bromodeoxyuridine (BrdU) at intervals from 12–48 h after [³H]TdR, to follow them from one cell cycle to the next. Labelling was revealed by combining [³H]TdR autoradiography with immunoperoxidase detection of BrdU in the same sections.
A single peak in the appearance of double-labelled cells was seen at 44 h, if [³H]TdR was given at 1200 h; following [3H]TdR at 0300 h, a peak of double labelling was seen at 48 h with the possibility of smaller peaks at 24 h and 36 h.
These results show that the 24 h periodicity in LI in this tissue is associated with a predominant cell cycle duration of 44–48 h, but that a few cells cycle more quickly. Double labelling with [³H]TdR and BrdU provides a useful method for establishing cell cycle duration by labelling S-phase cells in successive cell cycles.     

Cell cycle traverse in NHIK-3025 carcinoma of the uterine cervix after low-dose-rate irradiation

Theoretically, fractionation schemes could be tailored to the individual pattern of radiation-induced synchronization of cells in the radiosensitive G2 phase, leading to more effective radiotherapy. Using a human cervical carcinoma xenografted to nude mice, the effects of low-dose-rate irradiation on the cell cycle distribution were studied. Flow cytometric analysis demonstrated that cells accumulated in G2 + M phase 35 h after a total dose of 10 Gy of 137Cs irradiation. This accumulation time corresponded closely to the cell cycle time (Tc) (31 h) of this tumor, as determined by autoradiography. Further experiments are planned to determine the potential of fractionation schemes adjusted to the Tc-related accumulation in G2 in improving the effectiveness of radiotherapy.     

DNA-synthesizing cells in oral epithelium have a range of cell cycle durations: evidence from double-labelling studies using tritiated thymidine and bromodeoxyuridine

Mouse tongue epithelium is characterized by a circadian variation in the number of DNA-synthesizing cells (labelling index, LI). Cells undergoing DNA synthesis were labelled with tritiated thymidine [( 3H]TdR) at 0300 (peak LI) or 1200 h (low LI). The fate of these cells was assessed by injecting animals with bromodeoxyuridine (BrdU) at intervals from 12-48 h after [3H]TdR, to follow them from one cell cycle to the next. Labelling was revealed by combining [3H]TdR autoradiography with immunoperoxidase detection of BrdU in the same sections. A single peak in the appearance of double-labelled cells was seen at 44 h, if [3H]TdR was given at 1200 h; following [3H]TdR at 0300 h, a peak of double labelling was seen at 48 h with the possibility of smaller peaks at 24 h and 36 h. These results show that the 24 h periodicity in LI in this tissue is associated with a predominant cell cycle duration of 44-48 h, but that a few cells cycle more quickly. Double labelling with [3H]TdR and BrdU provides a useful method for establishing cell cycle duration by labelling S-phase cells in successive cell cycles.     

The acquisition of competence as a possible source of cell cycle variability in mouse fibroblasts

The kinetic analysis of the competence induction in Balb/c-3T3 cells shows that competence is acquired with a first order kinetics, whose rate depends on the concentration of the competence factor. Thus it is suggested that the competence induction may be a source of cell cycle variability for mouse fibroblasts.