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 ³ H-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°C so that cooling of selected cells in order to increase the yield would reduce the quality of the synchronized populations.     

The origin of variability in cell cycle durations of NHIK 3025 cells

The origin of cell cycle variability was investigated in NHIK 3025 cells synchronized by mitotic selection from an exponentially growing population. The variability in G1 durations was measured by flow cytometric analysis of the fraction of cells in G1 as a function of time after mitotic selection. Immediately before the first cells entered S, medium containing 2.0 mM thymidine was added to the cells, and removed when all the cells had reached S. Since the cells had approximately the same DNA content upon removal of the thymidine, the variability in the durations of S+G2+M was measured by counting the fraction of undivided cells as a function of time after removing the thymidine. Such a thymidine treatment did not affect the naturally occurring variability in cell cycle durations generated after the start of S. The results indicate that the cell cycle variability of NHIK 3025 cells can be adequately described by a cell cycle model consisting of at least two compartments, which the cells leave according to first order kinetics. The model accounts for the initial shoulder of the curve representing the fraction of undivided cells as a function of time after mitotic selection. Furthermore, it accounts for the reduction in the rate of entry into the subsequent cell cycle compared to the rate of entry into S. Both rate constants were equally reduced after serum stepdown.     

The role of protein metabolism in glucocorticoid-lnduced prolongation of G1 phase in human NHIK 3025 cells

It has previously been found that human NHIK 3025 cells have a glucocortiocoid-sensitive restriction point in mid-G1 phase of the cell cycle. When these cells were synchronized by mitotic selection and exposed to dexamethasone before the restriction point, G1 phase was prolonged whereas the rest of the cell cycle was unperturbed by the hormone. These observations were confirmed by flowcytometric mesurements of synchronized cells in the present study. Cells that received dexamethasone (10^?6 M) just after mitotic selection had a 4 hour prolongation of both G1 and the total cell cycle. However, the general rates of both protein synthesis and protein degradation were found not to be altered by the hormone, i.e., the rate of protein accumulation in dexamethasone exposed cells was equal to that of control cells. Dexamethasone exposed NHIK 3025 cells were found to be larger than control cells at the time of cell division. This is a direct consequence of a prolonged cell cycle duration with no change in general protein metabolism. It thus appears that the dexamethasone-induced prolongation of G1 phase is the result of a steroid-regulated G1 specific process(es) leading toward DNA replication, a process that does not alter general protein accumulation.     

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.     

Effect of serum step-down on protein metabolism and proliferation kinetics of NHIK 3025 cells

Human NHIK 3025 cells growing exponentially in 30% or 3% serum had population doubling times of 19.1 and 27.6 hours, respectively. These values were equal to the calculated protein doubling times (17.6 and 26.5 hours, respectively), showing that the cells were in balanced growth at both serum concentrations. Stepdown from 30% to 3% serum reduced the rate of protein synthesis within 1–2 hours, from 5.7% hour to 4.3% hour, while the rate of protein degradation was unchanged (1.7%/hour). In cells synchronized by mitotic selection from an exponentially growing population, the median cell cycle durations in 30% and 3% serum were 17.2 and 23.6 hours, respectively, which were also in good agreement with the protein doubling times. The median G₁ durations were 7.1 and 9.6 hours, respectively. Thus the duration of G₁ relative to the total cell cycle duration was the same in the two cases. Complete removal of serum for a period of 3 hours resulted in a 3-hour prolongation of the cell cycle regardless of the time after mitotic selection at which the serum was removed. For synchronized cells, the rate of entry into both the S phase and into the subsequent cell cycle were reduced in 3% serum as compared to 30% serum, the former rate being significantly greater than the latter at both serum concentrations. Our results thus indicate that these cells are continuously dependent upon serum throughout the entire cell cycle.     

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.     

Cell Kinetic Characteristics In Different Parts of Multicellular Spheroids of Human Origin

The growth fraction, the cell cycle time, and the duration of the individual cell cycle phases were determined as a function of distance from the surface of multicellular spheroids of the human cell line NHIK 3025. the techniques employed were percentage of labelled mitoses and labelling index measurements after autoradiography and flow cytometric measurements of DNA histograms. to separate cell populations from the different parts of the spheroid, fractionated trypsinization was employed. The results were compared with corresponding values in NHIK 3025 cell populations grown as monolayer cultures. While practically all cells in exponentially growing monolayer populations are proliferating, the growth fraction was between 0.6 and 0.7 in the outer parts of the spheroid. the inner region was mainly occupied by a necrotic mass. the proliferating fraction of the recognizable cells in the inner region was slightly below 0.5. the mean cell cycle time of NHIK 3025 cells in monolayer culture is 18 hr. the mean cell cycle time of proliferating cells in the periphery of the spheroid was 30 hr, compared to 41 hr in the inner region (150 μm from the spheroid surface). All phases of the cell cycle were prolonged compared to populations of exponentially growing monolayer cells. Within each part of the spheroid the distribution of cell cycle times was considerably broadened compared with monolayer populations.     

Doubling of cell mass is not necessary in order to achieve cell division in cultured human cells

When exponentially growing NHIK 3025 cells were shifted from medium containing 30% serum to medium containing 0.03% serum the rate of net protein accumulation was reduced due to both a reduction in the rate of protein synthesis and an increase in the rate of protein degradation. This change in growth conditions increased the protein doubling time from 18 to 140 h. The cell cycle duration of cells synchronized by mitotic selection was, however, only increased from 17 to 26 h by this treatment. Therefore, when the cells divide by the end of the first cell cycle following synchronization, the cells shifted to 0.03% serum contained far less protein than those growing continuously in 30% serum. Hence, the attainment of a critical cell mass is probably not controlling cell division for cells growing in a balanced state.     

Cell synchrony techniques. I. A comparison of methods

Selected cell synchrony techniques, as applied to asynchronous populations of Chinese hamster ovary (CHO) cells, have been compared. Aliquots from the same culture of exponentially growing cells were synchronized using mitotic selection, mitotic selection and hydroxyurea block, centrifugal elutriation, or an EPICS V cell sorter. Sorting of cells was achieved after staining cells with Hoechst 33258. After synchronization by the various methods the relative distribution of cells in G1, S, or G2 + M phases of the cell cycle was determined by flow cytometry. Fractions of synchronized cells obtained from each method were replated and allowed to progress through a second cell cycle. Mitotic selection gave rise to relatively pure and unperturbed early G1 phase cells. While cell synchrony rapidly dispersed with time, cells progressed through the cell cycle in 12 hr. Sorting with the EPICS V on the modal G1 peak yielded a relatively pure but heterogeneous G1 population (i.e. early to late G1). Again, synchrony dispersed with time, but cell-cycle progression required 14 hr. With centrifugal elutriation, several different cell populations synchronized throughout the cell cycle could be rapidly obtained with a purity comparable to mitotic selection and cell sorting. It was concluded that, either alone or in combination with blocking agents such as hydroxyurea, elutriation and mitotic selection were both excellent methods for synchronizing CHO cells. Cell sorting exhibited limitations in sample size and time required for synchronizing CHO cells. Its major advantage would be its ability to isolate cell populations unique with respect to selected cellular parameters.     

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.     

Selective detachment of mitotic cells by hypotonic salt solution

Summary A method has been developed to obtain synchronous populations from a human cell line which previously resisted the use of the selective harvest technique. A concentration of Colcemid was determined which reversibly enriched the mitotic population but avoided delays in cell cycle progression. Mitotic cells were then detached from monolayer cultures by brief treatment with hypotonic salt solutions. The resulting populations of line A244 were shown to be viable and syntchronous by following attachment efficiency and cycle time and by monitoring mitotic index and deoxyribonucleic acid synthesis. Hypotonic solutions offer no advantage in the selection of mitotic L-929 cells, a line commonly synchronized by selective harves. However, their use with both CV-1 and A244 cells provided large populations highly synchronized with respect to mitosis. This technique might be applied successfully to cell types which do not demonstrate a selective advantage at division.     

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.     

Cell Synchrony Techniques. I. A Comparison of Methods

Abstract Selected cell synchrony techniques, as applied to asynchronous populations of Chinese hamster ovary (CHO) cells, have been compared. Aliquots from the same culture of exponentially growing cells were synchronized using mitotic selection, mitotic selection and hydroxyurea block, centrifugal elutriation, or an EPICS V cell sorter. Sorting of cells was achieved after staining cells with Hoechst 33258. After synchronization by the various methods the relative distribution of cells in G₁ S, or G₂+ M phases of the cell cycle was determined by flow cytometry. Fractions of synchronized cells obtained from each method were replated and allowed to progress through a second cell cycle. Mitotic selection gave rise to relatively pure and unperturbed early G₁ phase cells. While cell synchrony rapidly dispersed with time, cells progressed through the cell cycle in 12 hr. Sorting with the EPICS V on the modal G₁ peak yielded a relatively pure but heterogeneous G₁ population (i.e. early to late G₁). Again, synchrony dispersed with time, but cell-cycle progression required 14 hr. With centrifugal elutriation, several different cell populations synchronized throughout the cell cycle could be rapidly obtained with a purity comparable to mitotic selection and cell sorting. It was concluded that, either alone or in combination with blocking agents such as hydroxyurea, elutriation and mitotic selection were both excellent methods for synchronizing CHO cells. Cell sorting exhibited limitations in sample size and time required for synchronizing CHO cells. Its major advantage would be its ability to isolate cell populations unique with respect to selected cellular parameters.     

Concentration-dependent effects of potassium dichromate on the cell cycle

Hexavalent chromium is found to be a strong mutagen, and it also is a potential carcinogen in man. DNA flow cytometry, growth measurements, and determinations of mitotic index show that 1-2 microM K2Cr2O7 produces a prolongation of the G2 phase of the cell cycle in NHIK 3025 cells. By increasing the chromate concentrations (greater than 2 microM K2Cr2O7) the cells are also arrested in G2 phase. We have found, using synchronized cells and measuring cell cycle time, that the most chromate-sensitive part of the cell cycle is S phase. This phase is also somewhat prolonged, and the cells became arrested in early S phase at high toxic K2Cr2O7 concentrations (8 microM). Our results thus indicate that K2Cr2O7 has an effect within S phase--maybe on DNA/RNA synthesis--and also interferes with processes necessary for progression through the G2 phase.     

Cell cycle progression in human cells following re-oxygenation after extreme hypoxia: consequences concerning initiation of DNA synthesis

Abstract. The initiation of DNA synthesis and further cell cycle progression in cells during and following exposure to extremely hypoxic conditions in either G₁ or G₂+M has been studied in human NHIK 3025 cells. Populations of cells, synchronized by mitotic selection, were rendered extremely hypoxic (< 4 p.p.m. O₂) for up to 24n h. Cell cycle progression was studied from flow cytometric DNA recordings. No accumulation of DNA was found to take place during extreme hypoxia. Cells initially in G₁ at the onset of treatment did not enter S during up to 24 h exposure to extreme hypoxia, but started DNA synthesis in a highly synchronous manner within 1.5 to 2.25 h after reoxygenation. The duration of S phase was only slightly affected (increased by ≅10%) by the hypoxic treatment. This suggests that the DNA synthesizing machinery either remains intact during hypoxia or is rapidly restored after reoxygenation. Cells initially in G₂ at the onset of hypoxia were able to complete mitosis, but further cell cycle progression was blocked in the subsequent G^ Following reoxygenation, these cells progressed into S phase, but the initiation of DNA synthesis was delayed for a period corresponding to at least the duration of normal G₁ and did not appear in a synchronous manner. In fact, cell cycle variability was found to be increased rather than decreased as a result of exposure to hypoxia starting in G₂. We interpret these findings as an indication that important steps in the preparation for initiation of DNA synthesis take place before mitosis. Furthermore, the change in cell cycle duration induced by hypoxia commencing in G₁ is of a nature other than that induced by hypoxia commencing in other parts of the cell cycle.     

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.     

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.     

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.     

Phase response versus positive and negative division delay in animal cells

The time of median cell division in V79 Chinese hamster cells following high serum pulses was determined for two synchronous cell generations following mitotic selection. Differences in cell cycle time for each pair of pulse and control cultures were computed and plotted as a function of time of serum pulse. This phase response curve for hamster cells with an 8.5 h cell cycle shows a characteristic biphasic pattern. Beginning 0.5 h after mitotic selection, pulses with serum produce delays in the midpoint of the subsequent mitotic waves. Delay is maximum at 1.5 h. Delays give way abruptly to advances at 2.5 h and the amount of advance then decreases as pulses are given between 3 and 5 h into the cycle. At 5 h decreasing advances become delays, with increasing delays due to serum pulses occurring between 5 and 6 h. Delays again give way abruptly to advances at 6 h and again the amount of advance decreases through the late portion of the cycle. Pulses very late in the cycle appear to generate phase delays. This biphasic response to serum is interpreted as an expression of an underlying time-keeping oscillator whose period is nominally of 4 h duration.     

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.