Cell cycle-dependent expression of surface antigens in murine T-lymphoma cells : II. Murine leukemia virus gp70 increases in S phase

Studies were undertaken to identify cell surface markers specific for different phases of the cell cycle. Antisera were prepared in rabbits against membrane protein preparations from synchronized BW 5147 cells, an AKR mouse T-lymphoma cell line, in the G1, S, G2 or M phases of the cell cycle. These antisera were used to precipitate radioiodinated surface proteins from synchronized cells in the different phases. The immunoprecipitates were quantitatively analyzed by sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Cells in S phase had significantly higher concentrations of proteins weighing 70 × 10³ and 165 × 10³ D than cells in G1 or G2 phase. The other major labeled surface components did not vary. These results were confirmed by quantitative absorption of the antisera with synchronized cells. Comparative analysis of the antisera showed that the 165 × 10³ D peak contained at least two antigens, one recognized by both a-G1 and a-S and the other by a-G1 only. Though cells in S phase had large quantities of the 70 × 10³ D protein, intact and SDS-solubilized membrane preparations from S phase could not elicit in rabbits any antibody against that protein. These antisera did, however, have good antibody titers to the other major protein peaks and the antisera developed against cells in G1, G2 or M had good anti-70 × 10³ activity. The results suggest a qualitative molecular change in the 70 × 10³ protein during S phase.     

Characterization of functionally active interleukin‐18/eGFP fusion protein expression during cell cycle phases in recombinant chicken DF1 Cells

The dependence of foreign gene expression on cell cycle phases in mammalian cells has been described. In this study, a DF1/chIL‐18a cell line that stably expresses the fusion protein chIL‐18 was constructed and the enhanced green fluorescence protein connected through a (G₄S)₃ linker sequence investigated the relationship between cell cycle phases and fusion protein production. DF1/chIL‐18a cells (1 × 10⁵) were inoculated in 60‐mm culture dishes containing 5 mL of media to achieve 50%–60% confluence and were cultured in the presence of the cycle‐specific inhibitors 10058‐F4, aphidicolin, and colchicine for 24 and 48 h. The percentage of cell density and mean fluorescence intensity in each cell cycle phase were assessed using flow cytometry. The inhibitors effectively arrested cell growth. The fusion protein production rate was higher in the S phase than in the G0/G1 and G2/M phases. When cell cycle progression was blocked in the G0/G1, S, and G2/M phases by the addition of 10058‐F4, aphidicolin, and colchicine, respectively, the aphidicolin‐induced single cells showed higher fusion protein levels than did the 10058‐F4‐ or colchicine‐induced phase cells and the uninduced control cells. Although the cells did not proliferate after the drug additions, the amount of total fusion protein accumulated in aphidicolin‐treated cells was similar to that in the untreated cultures. Fusion protein is biologically active because it induces IFN‐γ production in splenocyte cultures of chicken. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:581–591, 2016     

Ribosomal ribonucleic acid maturation in synchronous strain l cells

Summary The incorporation of [³H]-5-uridine into cytoplasmic 18S and 28S ribosomal ribonucleic acid (rRNA) was examined in Colcemid-synchronized strain L cells during G1 and S phases of the cell cycle in the presence of 5×10⁻⁵ m uridine, which was determined to be the saturating concentration for this system. The data show that in S phase a significant increase occurs in the level of [³H]-5-uridine incorporation into each rRNA species. During a 90-min exposure period, S phase cells incorporate 3.4 times as much [³H]-5-uridine into 18S rRNA and 1.9 times as much into 28S rRNA as do G1 cells. The time required for maturation of the ribosomal RNA species during G1 and during S phase is the same, with 18S rRNA appearing in the cytoplasm in 20 min and 28S rRNA in 40 min.     

Lack of effect of butyrate on S phase DNA synthesis despite presence of histone hyperacetylation

The effects of sodium butyrate on [³H]thymidine incorporation and cell growth characteristics in randomly growing and synchronized HeLa S₃ cells have been examined in an attempt to determine what effects, if any, butyrate has on S phase cells. Whereas 5 mM sodium butyrate rapidly inhibits [⁵H]thymidine incorporation in a randomly growing cell populations, it has no effect on incorporation during the S phase in cells synchronized by double thymidine block techniques. This lack of effect does not result from an impaired ability of the S phase cells to take up butyrate, since butyrate administration during this period leads to histone hyperacetylation that is identical with that seen with butyrate treatment of randomly growing cells. Furthermore, the ability to induce such hyperacetylation with butyrate during an apparently normal progression through S phase indicates that histone hyperacetylation probably has no effect on the overall process of DNA replication. Temporal patterns of [³H]thymidine incorporation and cell growth following release from a 24-h exposure to butyrate confirm blockage of cell growth in the G1 phase of the cell cycle. Thus, the inhibition by butyrate of [³H]thymidine incorporation in randomly growing HeLa S₃ cell populations can be accounted for solely on the basis of a G1 phase block, with no inhibitory effects on cells already engaged in DNA synthesis or cells beyond the G1 phase block at the time of butyrate administration.     

Expression of the mitochondrial genome in HeLa cells. XXI. Mitochondrial protein synthesis during the cell cycle

Chloramphenicol sensitive [³H]leucine incorporation into protein (due to mitochondrial protein synthesis) in synchronized HeLa cells has been found to continue throughout interphase, its rate per cell approximately doubling from the G₁ to the G₂ phase. This increase in the rate of [³H]leucine incorporation during the cycle does not seem to parallel closely the increase in cell mass. In fact, the observations made on cultures incubated at 34.5 °C, where the G₁ and S phases are better resolved than at 37 °C, indicate that the rate remains constant during the G₁ phase, and starts to accelerate with the onset of nuclear DNA synthesis. Correspondingly, on a per unit mass basis, there appears to be a slight decline in the rate of [³H]leucine incorporation into protein during the G₁ phase, which is compensated by an increase in the early S phase. No significant variations were observed in the mitochondrial leucine pool labeling during the cell cycle; therefore, the observed pattern of [³H]leucine incorporation into protein should reflect fairly accurately the behavior of mitochondrial protein synthesis. Evidence has been obtained indicating a depression in the rate of incorporation of [³H]leucine into protein in mitochondria of mitotic cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the products of mitochondrial protein synthesis has not revealed any differences in the size distribution of the proteins synthesized in the various portions of the cell cycle.     

Energy metabolism and ATP turnover time during the cell cycle of Ehrlich ascites tumour cells

The energy production in different parts of the cell cycle due to aerobic and aerobic glycolytic metabolism and ATP turnover time was estimated by measuring the oxygen consumption, lactate-pyruvate and ATP content of Ehrlich ascites tumour cells growing in vivo. Cell fractions of high purity from the various parts of the cell cycle were obtained by means of elutriator centrifuging. The total energy production for one cell cycle was estimated to be 19 × 10^?12 mol ATP, 60% of which was due to the aerobic metabolism. Whereas the total ATP production is unchanged during G1 a fairly exponential increase is found during the S and G2 + M phases. The total cellular ATP content increases from 12 fmol ATP at early G1 to 28 fmol ATP at G2 + M; this increase, however, is discontinuous and is most pronounced during G1 and during late S phase S phase/G2 + M. The ATP turnover time, as defined as the ratio between ATP content and ATP production, was found to increase significantly from 75 sec in early G1 to 120 sec in late G1 but was constantly 100 sec during the early, middle and late S phase as well as G2 + M. These variations indicate maximum energy-requiring processes during early G1 period of the cell cycle and are discussed in relation to K⁺Na⁺ flux and macromolecule synthesis.     

A Detailed Study of the Complex Cell Cycle of the Dinoflagellate Crypthecodinium cohnii Biecheler and Evidence for Variation in Histone H1 Kinase Activity

ABSTRACT By adding the protein synthesis inhibitor, emetine (10^-4 M) to a highly synchronized population of Crypthecodinium cohnii Biecheler 1938 at different phases of its cycle, we were able to determine: 1. The existence and the lengthening of the G2-Phase (30 min) in the first cycle (cycle with swimming G1 phase). 2. The time of the second cell cycle phases (cycle in the cyst): G1, 30 min; S, 1.5 h; G2, 2 h and M, 2 h. These results, together with the estimation of the cell volume of the two and four swimming daughter cells emerging from the cysts, allowed us to state the existence of two transition points: G1/S and G2/M, which are necessary for completion of mitosis. We completed this refined approach of the cell cycle in studying the activities of the histone H1 kinase either in dividing or in non-dividing Crypthecodinium cohnii cells with either total soluble proteins or the isolated mitotic kinase complex. The H1 kinase activity of this purified complex is noticeably higher (twice as high) in the dividing cells than in the non-dividing ones. These data are discussed in the light of the basic characteristics of the dinokaryon, and also compared with recent biochemical observations on the same organism and studies on other higher eukaryotic protists and metazoa.     

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.     

Mammalian cell fusion. VI. Regulation of mitosis in binucleate HeLa cells.

In two different cell fusion experiments a synchronized population of HeLa cells, prelabeled with ³H-TdR, was fused with an unlabeled one using inactivated Sendai virus. In the first experiment, HeLa cells in early G2 phase which were exposed to either 4 °C, cycloheximide, actinomycin D or X-irradiation were fused separately with untreated and more advanced G2 cells. A comparison of the rates of mitotic accumulation (in the presence of Colcemid) for the various classes of mono- and binucleate cells revealed that the hybrid (binucleate) cells were intermediate between those of the advanced and the retarded parental types indicating that the chromosome condensing factors of the advanced component were diluted as a result of such fusion. The manner in which the retarding effects of actinomycin D and cycloheximide were reversed in the hybrid cells suggested that proteins had a major role as chromosome condensing factors in the G2 mitotic transition. In the second experiment, when S phase HeLa cells were fused with those in G2, the resulting heterophasic (S/G2) binucleate cells reached mitosis at about the same time as the homophasic (S/S) cells of the lagging parent indicating a complete dominance of the S over the G2 with regard to their progress towards mitosis. However, the addition of Mg²⁺ (2 × 10^?2 M of MgCl₂) to the medium helped the G2 nuclei to enter mitosis asynchronously, which consequently induced premature chromosome condensation (PCC) in the S phase component. These data suggested that in the heterophasic (S/G2) binucleate cells the S phase component caused decondensation of the G2 chromatin thus blocking it from entering into mitosis. This effect which did not appear to be dose-dependent could be neutralized and the G2 nuclei relieved from this repression by an external supply of Mg²⁺ ions.     

Arrest of cell cycle progression of HeLa cells in the early G1 phase in K+-depleted conditions and its recovery upon addition of insulin and LDL

Cell cycle progression of synchronized HeLa cells was studied by measuring labeling of the nuclei with [³H]thymidine. The progression was arrested in a chemically defined medium in which K⁺ was replaced by Rb⁺ (Rb-CDM) but was restored upon addition of insulin and/or low density lipoprotein (LDL). Cells started DNA synthesis 12 hr after addition of insulin and/or LDL, regardless of the time of arrest, suggesting their arrest early in the G1 phase. After incubation of cells in Rb-CDM containing insulin or LDL singly for 3, 6, or 9 hr, replacement of the medium by that without an addition resulted in marked delay in entry of cells into the S phase, but in its replacement by medium containing both agents, the delay was insignificant. Synthesis of bulk protein, estimated as increase in the cell volume, was not strongly inhibited. From these results we conclude that cell cycle progression of HeLa cells in K^?-depleted CDM is arrested early in the G1 phase and that the arrest is due to lack of some protein(s) required for entry into the S phase that is synthesized in the early G1 phase.     

Cell cycle dependent expression of a glucose regulated cell surface glycoprotein

Summary A cell surface associated “glucose regulated protein” has been described on nontransformed human fibroblasts. To examine the distribution of that protein on human fibroblasts specific antisera were used. The antisera was used in conjunction with indirect immunofluorescence and revealed that the glucose regulated protein was present as fibers on spread cells. Further, the antisera was used in complement mediated cytotoxicity assays to examine cells during specific stages of the mitotic cell cycle. Fibroblasts were synchronized by serum starvation, hydroxyurea inhibition, or colcemid inhibition followed by mitotic selection. The results demonstrated that the glucose regulated protein was maximally displayed during the G₁ phase of the cell cycle and minimally displayed during the S and M phases. Research was supported by contract AG00697 from the National Institutes of Health.     

cis-Diamminedichloroplatinum-mediated crosslinking of nuclear proteins to DNA is cell cycle specific

Immunochemical analysis was employed to investigate the cell cycle-dependent protein-DNA crosslinking by cis-diamminedichloroplatinum II (cis-DDP), in HeLa-S3 cells. Cells synchronized by double thymidine block or hydroxyurea were released into S phase and incubated at 2-h intervals with cis-DDP as they progressed through S1, G2, M, and then into G1 and S phases of the subsequent cycle. Immunoblots of the DNA-crosslinked antigens reacted with antisera to 0.35 M NaCl extract or residue of HeLa S-phase nuclei revealed that several antigens changed their DNA-crosslinking pattern during the progression of HeLa cells through their reproductive cycle.     

Double minutes replicate once during S phase of the cell cycle

Double minutes (dm) characteristically exhibit greater numerical heterogeneity among tumor cells than do chromosomes. The biological basis of this heterogeneity was studied in human carcinoma cell line S 18. Pulse labeling of asynchronous cells with [³H]dThd, continuous labeling of synchronized cells with BrdUrd and prematurely condensed chromosome (PCC) studies of G1 and G2 phase S 18 cells indicate that dm-DNA replicates only once during S phase of the cell cycle. No evidence was found for replication of dm-DNA at G1 phase, G2 phase or mitotis. Cells observed at anaphase show imprecise distribution of dm to daughter cells. These studies suggest numerical heterogeneity of dm results from anomalous mitotic segregation rather than anomalous replication of dmDNA.     

Characteristics of the cell cycle of matrix cells in the mouse embryo during histogenesis of telencephalon

The cell cycle of matrix cells in the telencephalon of the mouse embryo at different stages at day 10, 13, and 17 of gestation was investigated by means of ³H-thymidine autoradiography.The cell cycle time of matrix cells in the day 10 group was found to be 7.0 h, and lengthened linearly with embryonic age. The cell cycle times of day 13 and 17 groups were 15.5 and 26.0 h, respectively.The duration of G1 and S phases also lengthened linearly with embryonic age. The durations of G1 phase were 0.1, 6.8, and 13.8 h, for day 10, 13, and 17 groups, respectively, and those of S phase were 5.1, 6.9, and 10.4 h, for day 10, 13, and 17 groups, respectively. On the other hand, the durations of both G2 and M phases remained unchanged and these were 1.0 and 0.8 h, respectively, throughout the embryonic stages.It was a characteristic of the alteration of the cell cycle of the telencephalon during mouse embryonic life that not only G1 but also S phases lengthened linearly with embryonic age and both G2 and M phases remained constant.     

The influence of the growth phase of enteric bacteria on electrotransformation with plasmid DNA

Salmonella typhimurium LB5000 andEscherichia coli JM109 were transformed by electroporation. In accordance with the chemical transformation methods, the growth phase of these electrocompetent bacteria had a strong impact on transformation efficiency. Survival of bacteria, after the high-voltage electrical pulse was also influenced by the growth phase. Both bacterial species were most successfully electrotransformed when microbial cells were harvested at the late lag phase. The second optimum for transformation reachedE. coli cells in the mid-exponential andS. typhimurium cells in the late exponential phase. Transformation efficiencies ranged from 3.4×10⁴ to 2.7×10⁵ transformants per μg DNA in the case ofS. typhimurium and from 2.8 × 10² to 8.8×10⁵ transformants per μg DNA in the case ofE. coli. Survival of cells after the electrical pulse in late lag and late exponential phases was about 20% higher than during other phases of growth. Preparing electrocompetent cells from later phases of their growth is more useful for practice, because it provides more biomass with good yield of transformants.     

The comparative cell cycle and metabolic effects of chemical treatments on root tip meristems. III. Chlorsulfuron

Intact and excised cultured pea roots (Pisum sativum L. cv Alaska) were treated with chlorsulfuron at concentrations ranging from 2.8 ×10^?4 M to 2.8×10^?6 M. At all concentrations this chemical was demonstrated to inhibit the progression of cells from G₂ to mitosis (M) and secondarily from G₁ to DNA synthesis (S). The S and M phases were not directly affected, but the transition steps into those phases were inhibited. Total protein synthesis was unaffected by treatment of intact roots with 2.8×10^?6 M chlorsulfuron. RNA synthesis was inhibited by 43% over a 24-h treatment period. It is hypothesized that chlorsulfuron inhibits cell cycle progression by blocking the G₂ and G₁ transition points through inhibition of cell cycle specific RNA synthesis.     

Differential effect of D-penicillamine on the cell kinetic parameters of various normal and transformed cellular types

The cell-growth-inhibitory and phase-specific effects of D-penicillamine on cell-cycle progression were investigated using cell-proliferation patterns, quantitative cell-cycle analysis by flow cytometry, and determination of the mitotic index and binucleate cell fraction of normal (rabbit articular chondrocytes, L 809, rabbit fibroblasts) and transformed (HeLa, L 929) cells. D-penicillamine treatment resulted in an inhibition of growth within a dose range of 5 × 10^?4 M to 7.5 × 10^?3 M. Examination of DNA by flow cytometric analysis revealed that rabbit articular chondrocytes were preferentially arrested in the G0/1 phase of the cell cycle, whereas the other cell lines were blocked in the G2 + M phase; the increase in the proportion of cells with G2 + M DNA content was partially due to an enhancement of binucleate cells, resulting in a cytokinesis perturbation for HeLa and L 929 cells. These results showed that D-penicillamine affects cell proliferation through different events according to cell type.     

Fluctuation of trypsin-like proteinase activity in the cell cycle of synchronized and growing HeLa cells, and the effect of GMCHA-OPhBut

HeLa cells synchronized by double-thymidine block were grown in Eagle's minimum essential medium supplemented with 10% calf serum, and the fluctuation of trypsin-like protease activity in the cell cycle was examined. Seven distinct activity peaks were observed in one cell cycle at a cell density of 2%: two peaks in S phase, one peak at the S/G2 boundary, one peak in early M phase and one at the M/G1 boundary, and two peaks in G1 phase. HeLa cells synchronized by a mitotic detachment technique also showed similar results at cell density of 4.8%. The appearance of trypsin-like proteinase activity in the cell cycle was markedly affected by cell density, and no definite peak was observed above 8%. trans-Guanidinomethylcyclohexanecarboxylic and 4-tert-butylphenyl ester (GMCHA-OPhBut), a specific inhibitor for trypsin and a strong inhibitor of HeLa cell growth, had no effect on the various events in the first S, G2 and M phases, such as the incorporation of [methyl-3H]thymidine into DNA, the increase in the cell concentration, and the appearance of trypsin-like proteinase activity, whereas it retarded the onset of the second S phase and the various events in the second S, G2 and M phases for 3 h. In particular, it induced the appearance of a new proteinase peak at the G1/S boundary.