Human DNA polymerase epsilon is expressed during cell proliferation in a manner characteristic of replicative DNA polymerases.

In order to shed light on the role of mammalian DNA polymerase epsilon we studied the expression of mRNA for the human enzyme during cell proliferation and during the cell cycle. Steady-state levels of mRNA encoding DNA polymerase epsilon were elevated dramatically when quiescent (G0) cells were stimulated to proliferate (G1/S) in a similar manner to those of DNA polymerase alpha. Message levels of DNA polymerase beta were unchanged in similar experiments. The concentration of immunoreactive DNA polymerase epsilon was also much higher in extracts from proliferating tissues than in those from non-proliferating or slowly proliferating tissues. The level of DNA polymerase epsilon mRNA in actively cycling cells synchronized with nocodazole and in cells fractionated by counterflow centrifugal elutriation showed weaker variation, being at its highest at the G1/S stage boundary. The results presented strongly suggest that mammalian DNA polymerase epsilon is involved in the replication of chromosomal DNA and/or in a repair process that may be substantially activated during the replication of chromosomal DNA. A hypothetical role for DNA polymerase epsilon in a repair process coupled to replication is discussed.     

A UV-responsive G2 checkpoint in rodent cells.

We have studied the effect of UV irradiation on the cell cycle progression of synchronized Chinese hamster ovary cells. Synchronization of cells in S or G2 phase was accomplished by the development of a novel protocol using mimosine, which blocks cell cycle progression at the G1/S boundary. After removal of mimosine, cells proceed synchronously through the S and G2 phases, allowing manipulation of cells at specific points in either phase. Synchronization of cells in G1 was achieved by release of cells after a period of serum starvation. Cells synchronized by these methods were UV irradiated at defined points in G1, S, and G2, and their subsequent progression through the cell cycle was monitored. UV irradiation of G1-synchronized cells caused a dose-dependent delay in entry into S phase. Irradiation of S-phase-synchronized cells inhibited progression through S phase and then resulted in accumulation of cells for a prolonged interval in G2. Apoptosis of a subpopulation of cells during this extended period was noted. UV irradiation of G2-synchronized cells caused a shorter G2 arrest. The arrest itself and its duration were dependent upon the timing (within G2 phase) of the irradiation and the UV dose, respectively. We have thus defined a previously undescribed (in mammalian cells) UV-responsive checkpoint in G2 phase. The implications of these findings with respect to DNA metabolism are discussed.     

Changes in membrane potential during the cell cycle

The membrane potential of isolated synchronized Chinese hamster lung cells (V79) has been determined as a function of their position in the cell cycle. During G 1 the cells exhibit a low but increasing membrane potential which rises sharply at the onset of the S phase. The elevated membrane potential is maintained throughout S and G 2 and declines again when the cells enter mitosis. Membrane potentials in an unsynchronized culture, which was recorded from both mitotic and interphase cells physically associated in groups and clusters, were similar to the plateau level obtained during S and G 2 in isolated synchronized cells, and exhibited little variation. It is concluded that although the membrane potential of isolated cells fluctuates during the cell cycle, it plays no causal role as a regulator of mitotic activity.     

Cell cycle dependence of cytotoxicity and clastogenicity induced by treatment of synchronized human diploid fibroblasts with sodium fluoride

To study the cell cycle dependence of cytotoxicity and clastogenicity of sodium fluoride (NaF), synchronized human diploid fibroblasts were treated with NaF during different phases of the cell cycle and analyzed. Exponentially growing cells were synchronized by the following two procedures. (1) The cells were synchronized at G₀/G₁ phase by a period of growth in medium containing 1% serum (low serum medium). (2) The cells were synchronized at the G₁/S boundary by growth in low serum medium, followed by hydroxyurea treatment (Tsutsui et al., 1984a). Synchronized cells were treated with NaF for 3 h during the G₁ phase or G₂ phase, and for each of three 3-h periods during the S phase which lasted 9 h. Cytotoxicity, as determined by a decrease in colony-forming ability, was dependent upon the phase of the cell cycle during which NaF treatment was administered. The highest lethality was induced in when the cultures were treated with NaF during the second or third 3 h of S phase (middle or late S phase, respectively), or G₂ phase. Little lethality was observed in cultures in G₁ phase. Inducibility of chromosome aberrations of the cells following treatment with NaF was also dependent upon the phase of the cell cycle. A significant increase in the incidence of chromosome aberrations was observed only in cultures treated with NaF during early and / or middle S phases of cell cycle. These results suggest that cytotoxicity and clastogenicity of NaF to cultured human diploid fibroblasts are cell cycle dependent, and that the cells in early and middle S phases are more sensitive to the effects.     

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.     

Sequential phsophorylation of histone subfractions in the Chinese hamster cell cycle.

Ion exchange chromatography and preparative electrophoresis were used to examine the phosphorylation of histone f1 and f3 subfractions in synchronized Chinese hamster cells (line CHO). Three discrete f1 phosphorylation events were demonstrated to occur in sequence during the cell cycle. The first event (f1G1) commenced in G1 2 hours prior to entry of cells into S phase; the second event (f1s) commenced simultaneously with initiation of DNA synthesis; and the third event (f1M) commenced when cells entered mitosis. F1M phosphorylation occurred simultaneously with the phosphorylation of histone f3 (which is not phosphorylated during G1, S, or G2). Fractionation of f1 and f3 revealed no differences in these sequential phosphorylation patterns among the various f1 and f3 subfractions, indicating that these phosphorylations are general biochemical events of the cell cycle. Phosphorylated (f1G1) was found to accumulate in cells as they traversed THEIR CELL CYCLE. F1s was phosphorylated to twice the extent of f1G1, but f1s did not accumulate in the cells as they passed through interphase. F1M was phosphorylated to about 4 times the extent of the first phosphorylated form (f1G1). A model of the relationship of histone phosphorylation to the cell cycle is presented which suggests that (a) f1G1 phosphorylation is involved with chromatin structural changes necessary for cell proliferation; (b) f1s phosphorylation is involved with DNA replication; (c) F1M and f3 phosphorylations are involved in chromosome condensation.     

Temperature-dependent dynamics of the villin headpiece helical subdomain,an unusually small thermostable protein

(15)N spin relaxation experiments were used to measure the temperature-dependence of protein backbone conformational fluctuations in the thermostable helical subdomain, HP36, of the F-actin-binding headpiece domain of chicken villin. HP36 is the smallest domain of a naturally occurring protein that folds cooperatively to a compact native state. Spin-lattice, spin-spin, and heteronuclear nuclear Overhauser effect relaxation data for backbone amide (15)N spins were collected at five temperatures in the range of 275-305 K. The data were analyzed using a model-free formalism to determine generalized order parameters, S, that describe the distribution of N-H bond vector orientations in a molecular reference frame. A novel parameter, Lambda=dln(1-S)/dln T is introduced to characterize the temperature-dependence of S. An average value of Lambda=4.5 is obtained for residues in helical conformations in HP36. This value of Lambda is not reproduced by model potential energy functions commonly used to parameterize S. The maximum entropy principle was used to derive a new model potential function that reproduces both S and Lambda. Contributions to the entropy, S(r), and heat capacity, C(r)(p), from reorientational conformational fluctuations were analyzed using this potential energy function. Values of S(r) show a qualitative dependence on S similar to that obtained for the diffusion-in-a-cone model; however, quantitative differences of up to 0.5k, in which k is the Boltzmann constant, are observed. Values of C(r)(p) approach zero for small values of S and approach k for large values of S; the largest values of C(r)(p) are predicted to occur for intermediate values of S. The results suggest that backbone dynamics, as probed by relaxation measurements, make very little contribution to the heat capacity difference between folded and unfolded states for HP36.     

Relationship Between Replication of Simian Virus 40 DNA and Specific Events of the Host Cell Cycle

The relationship between replication of simian virus 40 (SV40) DNA and the various periods of the host-cell cycle was investigated in synchronized CV(1) cells. Cells synchronized through a double excess thymidine procedure were infected with SV40 at the beginning or the middle of S, or in G(2). The first viral progeny DNA molecules were in all instances detected approximately 20 h after release from the thymidine block, independent of the time of infection. The length of the early, prereplicative phase of the virus growth cycle therefore depended upon the period of the cell cycle at which the cells were infected. Infection with SV40 was also performed on cells obtained in early G(1) through selective detachment of cells in metaphase. As long as the cells were in G(1) at the time of infection, the first viral progeny DNA molecules were detected during the S period immediately following, whereas if infection took place once the cells had entered S, no progeny DNA molecule could be detected until the S period of the next cell cycle. These results suggest that the infected cell has to pass through a critical stage situated in late G(1) or early S before SV40 DNA replication can eventually be initiated.     

DNA topoisomerase I and II activities during cell proliferation and the cell cycle in cultured mouse embryo fibroblast (C3H 10T1/2) cells

We have used C3H 10T1/2 cells to examine the regulation of topoisomerase activities during cell proliferation and the cell cycle. The specific activity of topoisomerase I was about 4-fold greater in proliferating (log phase) cells than in non-proliferating (confluent) cells. In synchronized cells, the bulk of the increased activity occurred during or just prior to S phase, depending upon the method of synchronization. A smaller increase in activity also occurred during G1 phase. The increase in activity during S phase was not altered by a hydroxyurea block at the G1/S phase boundary indicating that it is not directly coupled to DNA synthesis and is not the result of topoisomerase I gene dosage. The increase was inhibited by blocking cells at mid-G1 phase using isoleucine deprivation. Thus, the increase in activity during S phase is dependent on events occurring during mid- to late G1 phase. In contrast to the changes in topoisomerase I levels, the specific activity of topoisomerase II showed no detectable difference in proliferating vs non-proliferating cells. In addition, no detectable difference in topoisomerase II specific activity was seen in G1, S and M phases of the cell cycle. The differences in the activity profiles of the topoisomerases I and II during the cell cycle suggest that the two activities are regulated independently and may be required for different functions.     

Effect of tulipin on cell cycle progression analyzed by BrdUrd incorporation

The effect of tulipin, a protein from plant origin recently purified, on cell cycle progression has been analyzed in the sensitive EUE cells by BrdUrd incorporation. The cytofluorimetric results demonstrate that tulipin specifically interacts with the S phase, with a dose-dependent decrease of the total S phase cells and an increase of the G1/G2 cells after 4 h of treatment in the synchronized EUE cells, whereas in the asynchronous population it mainly causes a dose-dependent decrease in the incorporation of BrdUrd per cell.     

Hydroxyurea treatment affects the G1 phase in next generation CHO cells

DNA replication kinetics were studied in populations of synchronized CHO cells treated in the previous generation with hydroxyurea. These CHO cells were re-synchronized by selective detachment of mitotic cells after previously synchronized G1 traversing cultures were treated with 0.1 mM and 2 mM hydroxyurea for 9 and 13 h. Our results show that these cells exhibit a shortening of G1 of at least 1 h relative to cells selected in mitosis from untreated exponentially growing cultures. Survival studies indicated that the hydroxyurea treatments did not affect plating efficiencies. Cell viability was reduced when the initially synchronized populations were blocked with 2 mM, but not 0.1 mM hydroxyurea for greater than 13 h. DNA replication measurements after these blocks showed that all cultures treated with 2 mM hydroxyurea for either 9, 13 or 15 h were blocked at the same point near the G1/S boundary, and then progressed through S phase with similar kinetics. The observed shortening of G1 in the next generation of these cells was independent of both the concentration (0.1 or 2.0 mM) and the time (9 or 13 h) of the hydroxyurea block. These results suggest that specific events relating to the next cell generation can be uncoupled from DNA synthesis and can occur when hydroxyurea inhibits normal cell cycle traverse of G1 cells into and through S phase.     

Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system.

Conditional overexpression of human cyclins B1, D1, and E was accomplished by using a synthetic cDNA expression system based on the Escherichia coli tetracycline repressor. After induction of these cyclins in asynchronous Rat-1 fibroblasts, a decrease in the length of the G1 interval was observed for cyclins D1 and E, consistent with an acceleration of the G1/S phase transition. We observed, in addition, a compensatory lengthening of S phase and G2 so that the mean cell cycle length in populations constitutively expressing these cyclins was unchanged relative to those of their uninduced counterparts. We found that expression of cyclin B1 had no effect on cell cycle dynamics, despite elevated levels of cyclin B-associated histone H1 kinase activity. Induction of cyclins D1 and E also accelerated entry into S phase for synchronized cultures emerging from quiescence. However, whereas cyclin E exerted a greater effect than cyclin D1 in asynchronous cycling cells, cyclin D1 conferred a greater effect upon stimulation from quiescence, suggesting a specific role for cyclin D1 in the G0-to-G1 transition. Overexpression of cyclins did not prevent cells from entering into quiescence upon serum starvation, although a slight delay in attainment of quiescence was observed for cells expressing either cyclin D1 or cyclin E. These results suggest that cyclins D1 and E are rate-limiting activators of the G1-to-S phase transition and that cyclin D1 might play a specialized role in facilitating emergence from quiescence.     

Growth Conditions and Cell Cycle Phase Modulate Phase Transition Temperatures in RBL-2H3 Derived Plasma Membrane Vesicles

Giant plasma membrane vesicle (GPMV) isolated from a flask of RBL-2H3 cells appear uniform at physiological temperatures and contain coexisting liquid-ordered and liquid-disordered phases at low temperatures. While a single GPMV transitions between these two states at a well-defined temperature, there is significant vesicle-to-vesicle heterogeneity in a single preparation of cells, and average transition temperatures can vary significantly between preparations. In this study, we explore how GPMV transition temperatures depend on growth conditions, and find that average transition temperatures are negatively correlated with average cell density over 15°C in transition temperature and nearly three orders of magnitude in average surface density. In addition, average transition temperatures are reduced by close to 10°C when GPMVs are isolated from cells starved of serum overnight, and elevated transition temperatures are restored when serum-starved cells are incubated in serum-containing media for 12h. We also investigated variation in transition temperature of GPMVs isolated from cells synchronized at the G1/S border through a double Thymidine block and find that average transition temperatures are systematically higher in GPMVs produced from G1 or M phase cells than in GPMVs prepared from S or G1 phase cells. Reduced miscibility transition temperatures are also observed in GPMVs prepared from cells treated with TRAIL to induce apoptosis or sphingomyelinase, and in some cases a gel phase is observed at temperatures above the miscibility transition in these vesicles. We conclude that at least some variability in GPMV transition temperature arises from variation in the local density of cells and asynchrony of the cell cycle. It is hypothesized that GPMV transition temperatures are a proxy for the magnitude of lipid-mediated membrane heterogeneity in intact cell plasma membranes at growth temperatures. If so, these results suggest that cells tune their plasma membrane composition in order to control the magnitude of membrane heterogeneity in response to different growth conditions.     

Protein tyrosine nitration in the cell cycle

Nitration of tyrosine residues in proteins is associated with cell response to oxidative/nitrosative stress. Tyrosine nitration is relatively low abundant post-translational modification that may affect protein functions. Little is known about the extent of protein tyrosine nitration in cells during progression through the cell cycle. Here we report identification of proteins enriched for tyrosine nitration in cells synchronized in G0/G1, S or G2/M phases of the cell cycle. We identified 27 proteins in cells synchronized in G0/G1 phase, 37 proteins in S phase synchronized cells, and 12 proteins related to G2/M phase. Nineteen of the identified proteins were previously described as regulators of cell proliferation. Thus, our data indicate which tyrosine nitrated proteins may affect regulation of the cell cycle.     

Colcemid effects on B16 melanoma cell progression and aberrant mitotic division

Mitotic cells selectively harvested after several h of colcemid treatment are routinely used to obtain synchronized cell cultures. DNA flow cytometry shows that when colcemid-treated B16 mitotic cells divide, they give rise to daughter cells in G1, some of which contain abnormal amounts of DNA. Two subpopulations appear to exist, one having a DNA content distribution expected of G1 cells, another having a mean DNA content about 0.8 of expected and an SD of DNA content more than 5 times expected. The effect was dependent on dose and duration of exposure to colcemid. Colcemid was more cytotoxic to cells in G2 + M than to G1 + S phase cells, and it slowed the progression of G1 cells to S. These effects of colcemid were much greater in aneuploid B16 melanoma cells than in pseudodiploid Chinese hamster ovary (CHO) cells.     

Commitment to differentiation induced by retinoic acid in P19 embryonal carcinoma cells is cell cycle dependent

The rate at which P19 embryonal carcinoma cells in monolayer culture become anchorage dependent during differentiation induced by retinoic acid (RA) was investigated. In both nonsynchronized cultures and cultures synchronized by mitotic selection, the ability to grow in semisolid medium, characteristic of the malignant stem cell, decreased after a lag period of about 12 hr in the continuous presence of RA, prior to an increase in cell generation time. However, striking differences between synchronized and nonsynchronized cultures were observed in their commitment to differentiation following RA removal. After only 2 hr of exposure to RA, synchronized cells continued a program of differentiation in which they became anchorage dependent, while at least 24 hr of exposure was required for exponentially growing cells to become similarly committed. Induction of anchorage dependence by RA was also strikingly cell cycle dependent; 2 or 4 hr of exposure of synchronized cells to RA in G1 phase, when the intrinsic capacity for soft agar growth is low, was sufficient to commit cells to anchorage dependence, but a similar exposure in S phase was not. Together, these results suggested that interactions between cells in different cell cycle phases in asynchronous cultures influenced commitment since exposure to RA for more than one cycle (13 hr) was required for all cells to become anchorage dependent. Increased plasminogen activator secretion and epidermal growth factor binding, markers of certain differentiated cell types, increased only 3 and 5 days after RA addition, respectively, and were not induced by pulsed exposure to RA of less than 24 hr, even in synchronized cells.     

The cell cycle dependence of c-sis gene expression: artifactual conclusions in cells prepared by chemical but not physical techniques

The c-sis oncogene encoding the B-chain of platelet-derived growth factor (PDGF) may be involved in an autocrine growth stimulation of tumours expressing the PDGF receptor, such as glioblastomas and sarcomas. To investigate whether expression of c-sis RNA is regulated in a cell cycle dependent manner, human A172 glioblastoma cells were synchronized by either centrifugal elutriation or chemical blockage with the DNA synthesis inhibitors hydroxyurea or aphidicolin. In non-perturbed elutriated cells, c-sis RNA levels were lower in the S phase of the cell cycle than in the G1 phase. In contrast, the chemically synchronized cells revealed a transient rise in c-sis RNA shortly after drug release, in early S phase. The RNA changes occurring after release from drug inhibition represent cell recovery from drug induced metabolic disturbances rather than true cell cycle dependent effects.