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.     

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.     

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 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.     

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 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.     

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.     

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.     

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.     

Hypoxia-induced irreversible S-phase arrest involves down-regulation of cyclin A

We have studied hypoxia-induced cell cycle arrest in human cells where the retinoblastoma tumour suppressor protein (pRB) is either functional (T-47D cells) or abrogated by expression of the HPV18 E7 oncoprotein (NHIK 3025 cells). All cells in S phase are immediately arrested upon exposure to extreme hypoxia. During an 18-h extreme hypoxia regime, the cyclin A protein level is down-regulated in cells of both types when in S-phase, and, as we have previously shown, pRB re-binds in the nuclei of all T-47D cells (Amellem et al. 1996). Hence, pRB is not necessary for the down-regulation of cyclin A during hypoxia. However, our findings indicate that re-oxygenation cannot release pRB from its nuclear binding following this prolonged exposure. The result is permanent S-phase arrest even after re-oxygenation, and this is correlated with a complete and permanent down-regulation of cyclin A in the pRB functional T-47D cells. In contrast, both cell cycle arrest and cyclin A down-regulation in S phase are reversed upon re-oxygenation in non-pRB-functional NHIK 3025 cells after prolonged exposure to extreme hypoxia. Our results indicate that pRB is involved in permanent S-phase arrest and down-regulation of cyclin A after extreme hypoxia.     

Retinoic acid induces a specific membrane glycoprotein in human epithelial cell lines

Retinoic acid (RA) inhibits growth, increases the cytokeratin content, and alters the cytoskeleton of the human cervical cell line NHIK 3025. Using RA-treated NHIK 3025 cells as immunogen we prepared murine monoclonal antibodies (IgG1) which recognized an RA-induced cell-surface antigen which could not be detected in untreated NHIK 3025 cells. Analysis of the Triton soluble proteins by SDS-gel electrophoresis and immunoblotting revealed that the cell-surface antigen is a 140-kDa glycoprotein (gp140). gp140 was also shown to be induced by RA in HeLa S3 cells and constitutively expressed in the human trophoblast cell line BeWo. gp140 was also detected in other human epithelial cell lines, but not in human hematopoietic cells. Expression of gp140 was induced in HeLa S3 cells by nanomolar concentrations of RA, and in NHIK 3025 cells by micromolar amounts (1-10 microM). The glycoprotein was detectable 3-6 h following exposure to RA and its expression was reversible upon removal of RA from the medium. Our results indicate that gp140 is a newly identified RA-inducible epithelial membrane glycoprotein which may represent a phenotypic differentiation marker for epithelial cells.     

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.     

Counteraction of pRb-dependent protection after extreme hypoxia by elevated ribonucleotide reductase

We have studied hypoxia-induced cell cycle arrest in human cells where the retinoblastoma tumour suppressor protein (pRb) is either functional (T-47D and T-47DHU-res cells) or abrogated by expression of the HPV18 E7 oncoprotein (NHIK 3025 cells). We have previously found that pRb is dephosphorylated and rebound in the nucleus in T-47D cells arrested in S-phase during hypoxia and that this binding is protracted even following re-oxygenation. In the present study, however, we show that the long-lasting arrest following re-oxygenation induced by pRb-binding in the cell nuclei may be overruled by an elevated level of ribonucleotide reductase (RNR). This seems to create a forced DNA-synthesis, uncoordinated with cell division, which induces endoreduplication of the DNA. The data indicate that the cells initiating endoreduplication continue DNA-synthesis until all DNA is replicated once and then may start cycling and cell division with a doubled DNA-content. Corresponding data on the pRb-incompetent NHIK 3025-cells show similar endoreduplication in these. Thus, the data indicate that endoreduplication of DNA following re-oxygenation may come, either as a result of hypoxic arrest of DNA-synthesis when pRb-function is absent in the cells, or if it is overruled by increased RNR. The present study further shows that pRb not only protects the culture by arresting most of the cells that are exposed to extreme hypoxia in S-phase, but also increases cell survival by means of increased clonogenic ability of these cells. Interestingly, however, cells having an elevated level of RNR have equally high survival as wild-type cells following 20 h extreme hypoxia. If RNR-overruling of pRb-mediated arrest following re-oxygenation results in an unstable genome, this may therefore represent a danger of oncogenic selection as the protective effect of pRb on cell survival seems to be maintained.     

Phosphorylation-dephosphorylation of retinoblastoma protein not necessary for passage through the mammalian cell division cycle

Phosphorylation of the retinoblastoma protein (Rb) during the G1-phase of the mammalian cell division cycle is currently believed to be a controlling element regulating the passage of cells into S-phase. We find, however, that the suspension-grown cell lines U937, L1210, and MOLT-4 contain exclusively hyperphosphorylated Rb. Furthermore, when adherent NIH3T3 cells are grown at very low densities to avoid overgrowth and contact inhibition, they also contain only hyperphosphorylated Rb. NIH3T3 cells exhibit hypophosphorylation when the cells are grown at moderate to high cell densities. We propose that cultures of adherent cells such as NIH3T3, when grown to moderate cell densities, are made up of two populations of cells: (a) cells that are relatively isolated and therefore growing exponentially without contact inhibition, and (b) cells that are growth-inhibited by local cell density or contact inhibition. The common observation in adherent cell lines, that Rb is both hyper- and hypophosphorylated in the G1-phase and only hyperphosphorylated in the S- and G2-phases, is explained by the effects of cell density and contact inhibition. Thus, phosphorylation-dephosphorylation of Rb protein during the G1 phase is not a necessary process during the NIH3T3, L1210, MOLT-4, and U937 division cycles. We propose that phosphorylation-dephosphorylation of Rb is independent of the division cycle and is primarily determined by growth conditions throughout the 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.     

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.     

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.     

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.     

Kinetics of protein and DNA synthesis studied by mathematical modelling of flow cytometric protein and DNA histograms

Abstract. Mathematical models for histograms of cellular protein content as measured by flow cytometry were developed, based on theoretical protein distributions. These were derived from the age distribution of cells and the accumulation function for cellular protein content as a function of age within the cell cycle. A model assuming an exponential age distribution and an exponential protein. accumulation function was found to give the best representation of protein histograms of exponentially growing NHIK 3025 cells. This is in good agreement with the known kinetic behaviour of such cells. By the combined use of the protein histogram model and a similar model for DNA content, and assuming linear DNA accumulation during S, the fraction of cells in S, as a function of cellular protein content, was simulated. This function showed good agreement with values of the [³H]TdR labelling index scored in cells sorted by flow cytometry from 5-channel intervals of the protein histogram. The protein and DNA histogram models were combined into a two-dimensional model for correlated protein/DNA measurements. Comparison between simulated data and experimentally derived two-dimensional protein/DNA histograms gave further support to the cell kinetic assumptions underlying the models, but also identified some minor deviations which could not be recognized in the analysis of the one-dimensional histograms.     

G1/S control of anchorage-independent growth in the fibroblast cell cycle

We have developed methodology to identify the block to anchorage-independent growth and position it within the fibroblast cell cycle. Results with NRK fibroblasts show that mitogen stimulation of the G0/G1 transition and G1-associated increases in cell size are minimally affected by loss of cell anchorage. In contrast, the induction of G1/S cell cycle genes and DNA synthesis is markedly inhibited when anchorage is blocked. Moreover, we demonstrate that the anchorage-dependent transition maps to late G1 and shortly before activation of the G1/S p34cdc2-like kinase. The G1/S block was also detectable in NIH-3T3 cells. Our results: (a) distinguish control of cell cycle progression by growth factors and anchorage; (b) indicate that anchorage mediates G1/S control in fibroblasts; and (c) identify a physiologic circumstance in which the phenotype of mammalian cell cycle arrest would closely resemble Saccharomyces cerevisiae START. The close correlation between anchorage independence in vitro and tumorigenicity in vivo emphasizes the key regulatory role for G1/S control in mammalian cells.