Progressive Increase In Polyamine Levels In 9l Cells in vitro During the Cell Cycle: Comparison Between Cells Isolated By Centrifugal Elutriation and Cells Grown In Synchrony

Centrifugal elutriation was used to separate 9L rat brain tumour cells into fractions enriched in the G₁, S, or G₂/M phases of the cell cycle. Cells enriched in early G₁, phase were recultured, grown in synchrony, and harvested periodically for analysis of their DNA distribution and polyamine content. Mathematical analysis of the DNA distributions indicated that excellent synchrony was obtained with low dissersion throughout the cell cycle. Polyamine accumulation began at the time of seeding, and intracellular levels of putrescine, spermidine, and spermine increased continuously during the cell cycle. In cells in the G₂/M phase of the cell cycle, putrescine and spermidine levels were twice as high as in cells in the G₁, phase. DNA distribution and polyamine levels were also analysed in cells taken directly from the various elutriation fractions enriched in G₁, S, or G₂/M. Because we did not obtain pure S or G₂/M populations by elutriation or by harvesting synchronized cells, a mathematical procedure—which assumed that the measured polyamine levels for any population were linearly related to the fraction of cells in the G₁, S, and G₂/M phases times the polyamine levels in these phases and that polyamine levels did not vary within these phases—was used to estimate ‘true’ phase-specific polyamine levels (levels to be expected if perfect synchrony were achieved). Estimated ‘true’ phase-specific polyamine levels calculated from the data obtained from cells either sorted by elutriation or obtained from synchronously growing cultures were very similar.     

The differentiation of T-lymphocytes. III. The behaviour of subpopulations of mouse thymus cells in short-term cell culture.

Seven human cultured lymphoblastoid cell lines (CLL) were divided into two major groups based on studies of their cell cycle characteristics and surface Ig. CLL I (lines CL, MW, HH and TM) had generation times ranging from 25–40 hr, S phase times of 10–12 hr, G₂ + M times of 6–8 hr, and demonstrated sharp differences between the percentage of SIg(+) cells in different phases of the cell cycle. Line TM was particularly discordant with the highest percentage of SIg(+) cells in G₂ + M. CLL II (lines PS, JR and HT) demonstrated generation times ranging from 18–21 hr, S phase times of 7–10 hr and G₂ + M phase times of 2 hr. In this second group, two of the three CLLs had no differences between cells taken from different points of the cell cycle. DNA synthesis and cell density could not be correlated with either of the above major parameters, i.e. cell cycle times or SIg expression. The results suggest that human CLLs fall into subgroups in which specific patterns of cellular and immune functions may predominate.     

Dynamics of association of origins of DNA replication with the nuclear matrix during the cell cycle

DNA of replication foci attached to the nuclear matrix was isolated from Chinese hamster ovary cells and human HeLa cells synchronized at different stages of the G₁ and S phases of the cell cycle. The abundance of sequences from dihydrofolate reductase ori-β and the β-globin replicator was determined in matrix-attached DNA. The results show that matrix-attached DNA isolated from cells in late G₁ phase was enriched in origin sequences in comparison with matrix-attached DNA from early G₁ phase cells. The concentration of the early firing ori-β in DNA attached to the matrix decreased in early S phase, while the late firing β-globin origin remained attached until late S phase. We conclude that replication origins associate with the nuclear matrix in late G₁ phase and dissociate after initiation of DNA replication in S phase.     

CIRCADIAN RHYTHMS IN MOUSE EPIDERMAL BASAL CELL PROLIFERATION

Several kinetic parameters of basal cell proliferation in hairless mouse epidermis were studied, and all parameters clearly showed circadian fluctuations during two successive 24 hr periods. Mitotic indices and the mitotic rate were studied in histological sections; the proportions of cells with S and G₂ phase DNA content were measured by flow cytometry of isolated basal cells, and the [³H]TdR labelling indices and grain densities were determined by autoradiography in smears from basal cell suspensions. The influx and efflux of cells from each cell cycle phase were calculated from sinusoidal curves adapted to the cell kinetic findings and the phase durations were determined. A peak of cells in S phase was observed around midnight, and a cohort of partially synchronized cells passed from the S phase to the G₂ phase and traversed the G₂ phase and mitosis in the early morning. The fluctuations in the influx of cells into the S phase were small compared with the variations in efflux from the S phase and the flux through the subsequent cell cycle phases. The resulting delay in cell cycle traverse through S phase before midnight could well account for the accumulation of cells in S phase and, therefore, also the subsequent partial synchrony of cell cycle traverse through the G₂ phase and mitosis. Circadian variations in the duration of the S phase, the G₂ phase and mitosis were clearly demonstrated.     

REGENERATIVE PROLIFERATION OF MOUSE EPIDERMAL CELLS FOLLOWING ADHESIVE TAPE STRIPPING

The proliferating cells of mouse epidermis (basal cells) can be separated from the non-proliferating cells (differentiating cells) (Laerum, 1969) and brought into a mono-disperse suspension. This makes it possible to determine the cell cycle distributions (e.g. the relative number of cells in the G^ S and (G₂+ M) phases of the cell cycle) of the basal cell population by means of micro-flow fluorometry. To study the regenerative cell proliferation in epidermis in more detail, changes in cell cycle distributions were observed by means of micro-flow fluorometry during the first 48 hr following adhesive tape stripping. ³H-TdR uptake (LI and grain count distribution) and mitotic rate (colcemid method) were also observed. An initial accumulation of G₂ cells was observed 2 hr after stripping, followed by a subsequent decrease to less than half the control level. This was followed by an increase of cells entering mitosis from an initial depression to a first peak between 5 and 9 hr which could be satisfactorily explained by the changes in the G₂ pool. After an initial depression of the S phase parameters, three peaks with intervals of about 12 hr followed. The cells in these peaks could be followed as cohorts through the G₂ phase and mitosis, indicating a partial synchrony of cell cycle passage, with a shortening of the mean generation time of basal cells from 83-3 hr to about 12 hr. The oscillations of the proportion of cells in G₂ phase indicated a rapid passage through this cell cycle phase. The S phase duration was within the normal range but showed a moderate decrease and the Gj phase duration was decreased to a minimum. In rapidly proliferating epidermis there was a good correlation between change in the number of labelled cells and cells with S phase DNA content. This shows that micro-flow fluorometry is a rapid method for the study of cell kinetics in a perturbed cell system in vivo.     

Macrophage-mediated cytostatic activity blocks lymphoblast cell cycle progression independently in both G1 phase and S phase

Recent work has shown that macrophage-mediated cytostatic activity inhibits cell cycle traverse in G1 and/or S phase of the cell cycle without affecting late S, G2, or M phases. The present report is directed at distinguishing between such cytostatic effects on G1 phase or S phase using the accumulation of DNA polymerase alpha as a marker of G1 to S phase transition. Quiescent lymphocytes stimulated with concanavalin A undergo a semisynchronous progression from G0 to G1 to S phase with a dramatic increase in DNA polymerase alpha activity between 20 and 30 hr after stimulation. This increase in enzyme activity was inhibited, as was the accumulation of DNA, when such cells were cocultured with activated murine peritoneal macrophages during this time interval. However, if mitogen-stimulated lymphocytes were enriched for S-phase cells by centrifugal elutriation and cocultured with activated macrophages for 4-6 hr, DNA synthesis was inhibited but the already elevated DNA-polymerase activity was unaffected. Similar results were obtained when a virally transformed lymphoma cell line was substituted as the target cell in this assay. These results show that both G1 and S phase of the cycle are inhibited and suggest that inhibition of progression through the different phases may be accomplished by at least two distinct mechanisms.     

Arrest of the cell cycle reduces susceptibility of target cells to perforin-mediated lysis

Cytotoxic T lymphocytes secrete a pore-forming cytolysin, perforin, that damages membranes of target cells. They also ligate Fas receptors on target cells and provoke apoptotic death. A20 (B lymphoma) and P815 (mastocytoma) cell lines were examined for their susceptibility to perforin-mediated lysis and to Fas-induced apoptosis after blockade of the cell cycle at the G₁/S interface. Cells were arrested at the G₁/S interface by inhibition of DNA synthesis with thymidine or aphidicolin. Subsequently, the treated cells were incubated either with CTL cytotoxic granules or the Fas-specific monoclonal antibody Jo-2. We show that arrest of the cell cycle at the G₁/S interface markedly reduced the susceptibility of target cells to perforin-mediated lysis. In contrast, growth arrest with thymidine or aphidicolin increased susceptibility of A20 and P815 cells to Fas-mediated apoptosis. Susceptibility to lysis by intact CTLs was not affected significantly by blockade of target cells with aphidicolin or thymidine. When cells surviving exposure to perforin-containing granules were isolated on Ficoll density gradients and cell-cycle profiles were examined by flow cytometry, the ratio of G₁ to G₂cells increased among the survivors exposed to granules in contrast to controls incubated with buffer alone. The data suggest that cells in G₁ phase of the cell cycle are less susceptible to the perforin pathway than cells in G₂and S phases but are more susceptible to the Fas pathway. J. Cell. Biochem. 69:425–435, 1998. © 1998 Wiley-Liss, Inc.     

Calmodulin and cell proliferation

The calmodulin content of synchronized Chinese hamster ovary (CHO-K₁) cells was determined at each phase of the cell cycle. The calmodulin content was minimum in the G₁ phase, increased after the cells entered S phase and reached the maximum level at the late G₂ or early M phase. When 30 μM of W-7 (calmodulin antagonist) was added at the S phase, the cell cycle was blocked at the late G₂ or early M phase. The addition of W-7 also prevented the morphological changes caused by cholera toxin. These results suggest that calmodulin plays an important role in the phases through S to M, possibly in the initiation of DNA synthesis and in the mitosis.     

Cell cycle analysis and synchronization of the TN-368 insect cell line

Summary A cell cycle analysis of theTrichoplusia ni (TN-368) insect cell line is described. By means of autoradiography and percent labeled metaphase data, the cell cycle parameters were determined to be as follows: S, 4.5 hr; G₂, 8.5 hr; M, 0.5 hr; G₁, 1.0 hr; the total cell time being 14.5 hr. A synchronization procedure using 50mm thymidine in a double block procedure was used to provide a method of obtaining a large number of cells in particular cell cycle phases, especially S and G₂. This work was supported in part by U.S. Environmental Protection Agency Grant R-802516.     

REGENERATIVE PROLIFERATION OF MOUSE EPIDERMAL CELLS FOLLOWING APPLICATION OF A SKIN IRRITANT (CANTHARIDIN)

The strong skin irritant cantharidin dissolved in benzene was applied to the back of hairless mice. Single cell suspensions of epidermal basal cells were obtained and flow microfluorometric measurements of cellular DNA content were made. Smears were made for autoradiography, and the [³H]TdR labelling index (LI) and mean grain count (MGC) were assessed up to 3 days after cantharidin application. Three successive peaks of cells with S phase DNA content accompanied by three LI peaks were observed. The first two peaks were follwed by peaks of cells in G₂ phase, indicating that after the acute cell injury caused by cantharidin the cells traversed the cell cycle in partial synchrony through two subsequent cell cycles, each of 10–12 hr duration. During this phase of rapid proliferation the LI reached the proportion of cells in S phase, contrary to what is observed in untreated mouse epidermis, where the labelled cells contribute to about half the proportion of cells with S phase DNA content. The first two peaks of cells in S phase and LI coincided with an increased MGC, whereas the third peak was accompanied by a MGC significantly below control values. This indicates that this latter peak is due to a longer DNA synthesis time rather than to a partially synchronized and increased cell proliferation. The duration of the G₁, S and G₂ phases seems to be reduced initially in rapidly proliferating epidermis.     

Analysis of differential sensitivities of synchronized HeLa S3 cells to radiations and chemical carcinogen during the cell cycle. II. Ultraviolet light

UV-induction of thymine dimers in cellular DNA and their excision during different phases of the cell cycle of HeLa S3 cells were studied. Induction of thymine dimers was higher in the mitotic phase and the middle of the S phase than in the G₁ phase and from the late S phase to the early G₂ phase which are rather insensitive to UV. However, there is no significant difference in excision rate of UV-induced thymine dimers from the irradiated cells through the cell cycle. These findings indicate that the cyclic variation of UV-survivals during the cell cycle may be due to differences in the amount of thymine dimers in cellular DNA induced by UV-irradiation.     

Effect of staurosporine on MOLT-4 cell progression through G2 and on cytokinesis

Staurosporine (SSP) is an inhibitor of a variety of protein kinases with an especially high affinity towards protein kinase C. Whereas SSP has been shown to halt the cell cycle progression of various normal, nontransformed cell types in G₁, most virus transformed or tumor cells are unaffected in G₁ but arrest in G₂ phase. SSP has also been observed to increase the appearance of cells with higher DNA content, suggestive of endoreduplication, in cultures of tumor cells. Using multivariate flow cytometry (DNA content vs. expression of cyclin B, nucleolar p120 protein, or protein reactive with Ki-67 antibody) which makes it possible to discriminate cells with identical DNA content but at different phases of the cycle, we have studied the cell cycle progression of human lymphocytic leukemic MOLT-4 cells in the presence of 0.1 μM SSP.MOLT-4 cells did not arrest in G₁ or G₂ phase in the presence of the inhibitor. Rather, they failed to undergo cytokinesis, entering G₁ phase at higher DNA ploidy (tetraploidy; G_1T), and then progressed through S_T (rereplication) into G_2T and M_T. The rates of entrance to G₂ and G_2T were essentially identical, indicating that the rates of cell progression through S and S_T as well as through G₂ and G_2T, respectively, were similar. Cells entrance to mitosis and mitotic chromatin condensation were also similar at the diploid and tetraploid DNA content level and were unaffected by 0.1 μM SSP. No evidence of growth imbalance (altered protein or RNA to DNA ratio) was observed in the case of tetraploid cells. The data show that, in the case of MOLT-4 cells, all events associated with the chromosome or DNA cycle were unaffected by SSP; the only target of the inhibitor appears to be kinase(s) controlling cytokinesis. © 1994 Wiley-Liss, Inc.     

Synchronization of drosophila cells in culture

Summary We synchronized Drosophila cell lines (Schneider's line 2 and K_c) by allowing the cells to enter the stationary phase of growth and then diluting them into fresh culture medium. The cells of both cell lines entered S phase, after an 8- to 14-hr delay, in a state of partial synchrony; 60 to 80% of the cell population accumulated in S phase. Measurements of the cell cycle phases of Schneider's line 2 cells (S=14 to 16 hr; G₂=6 to 8 hr; M=0.4 hr) were similar to those of K_c cells. This work was performed under the auspices of the U.S. Energy Research and Development Administration. A.R. was supported by an NIH post-doctoral fellowship, No. CA01060.     

STUDIES ON THE DIVISION CYCLE OF MAMMALIAN CELLS : V. Modifications of Time Parameters by Different Steady-State Culture Conditions

In cultures of murine neoplastic mast cells, the duration of different phases of the division cycle (G₁, S, G₂, and mitosis [M]) was determined under optimal and several well-defined suboptimal growth conditions. Two methods of evaluation were applied to the same culture system: first, the relative number of G₁, S, G₂, and M cells was determined by pulse labeling of samples with thymidine-³H and subsequent radioautography in conjunction with a microfluorometric technique permitting rapid measurements of cellular DNA content; second, after pulse labeling with thymidine-³H, the variations with time of the mitotic labeling index were analyzed. Suboptimal culture conditions were obtained by reducing the concentration of single essential medium components (leucine, glucose, or serum) or by the addition of specific metabolic inhibitors (actinomycin D, amethopterin). Growth-limiting culture conditions resulted in increased generation times. Even under control conditions, the cell number doubling time exceeded the generation time, and this difference was more pronounced in suboptimal media. Under most of the suboptimal conditions tested, the increase in generation time was attributable primarily to an extended duration of the G₁ phase. Under certain growth-limiting conditions, however, other phases were also prolonged. In addition, the variabilities of the generation time and of certain cell cycle phases were increased under suboptimal culture conditions. Results obtained by the two methods of evaluation were, in general, in good agreement with each other. Some differences were, however, observed and interpreted in terms of cell death and/or asymmetric frequency distributions of cell cycle parameters.     

DNA synthesis and the cell cycle in cultures of normal and mutant (mdg/mdg) embryonic mouse muscle cells.

The relationship between cell fusion, DNA synthesis and the cell cycle in cultured embryonic normal and dysgenic ($\text{mdg}\text{mdg}$) mouse muscle cells has been determined by autoradiography. The experimental evidence shows that the homozygous mutant myotubes form by a process of cell fusion and that nuclei within the myotubes do not synthesize DNA or undergo mitotic or amitotic division. The duration of the total cell cycle and its component phases was statistically the same in 2-day normal and mutant ($\text{mdg}\text{mdg}$) myogenic cultures with the approximate values: T, 21.5 hr; G₁, 10.5 hr; S, 7.5 hr; and G₂, 2.5 hr. In both kinds of cultures, labeled nuclei appeared in myotubes 15–16 hr after mononucleated cells were exposed to [³H]thymidine, and the rate of incorporation of labeled nuclei into multinucleated muscle cells was comparable in control and dysgenic cultures. Thus, homozygous $\text{mdg}\text{mdg}$ muscle cells in culture are similar to control cells with respect to their mechanism of myotube formation and the coordinate regulation of DNA synthesis and the cell cycle during myogenesis.     

Synchronization of the Human Promyelocytic Cell Line HL 60 By Thymidine

Cultures of the promyelocytic cell line HL 60 were synchronized with thymidine. A concentration of 0.05 mM thymidine and an exposure time of 24 hr was found optimal for blocking about 90% of the cells in S phase. Following release from the thymidine block the cell cultures were followed intermittently over 40 hr for fluctuation in cell numbers, labelling with radioactive thymidine and nuclear DNA distributions. Mathematical evaluation of the results revealed a cycling time of 18.6 hr and a duration of specific cell phases of 8.6 hr, 7.1 hr and 2.9 hr for G₁, S and G₂+ M, respectively. the doubling time was 26 hr and the growth fraction was estimated as 1.     

Conservative homologous recombination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells

DNA double-strand breaks (DSBs) are repaired by either homologous recombination (HR) or non-homologous end joining (NHEJ) in mammalian cells. Repair with NHEJ or HR using single-strand annealing (SSA) often results in deletions and is generally referred to as non-conservative recombination. Error-free, conservative HR involves strand invasion and requires a homologous DNA template, and therefore it is generally believed that this type of repair occurs preferentially in the late S, G₂ and M phases of the cell cycle, when the sister chromatid is available. There are several observations supporting this hypothesis, although it has not been tested directly. Here, we synchronize human SW480SN.3 cells in the G₁/G₀ (with serum starvation), S (with thymidine block) and M (with nocodazole) phases of the cell cycle and investigate the efficiency of conservative HR repair of an I-SceI-induced DSB. The frequency of HR repair of DSBs was 39 times higher in S-phase cells than in M-phase cells and 24-fold higher than in G₁/G₀ cells. This low level of conservative HR occurs even though a homologous template is present within the recombination substrate. We propose that this can be explained by an absence of recombination proteins outside the S phase or alternatively that there maybe factors that suppress HR in G₁/G₀ and M. Furthermore, we found that HR repair of DSBs involves short tract gene conversion in all the phases of the cell cycle. This indicates that the same pathway for conservative HR is employed in the repair of DSBs regardless of phase of the cell cycle and that only the frequency is affected.     

Invading cancer cells are predominantly in G0/G1 resulting in chemoresistance demonstrated by real-time FUCCI imaging

Invasive cancer cells are a critical target in order to prevent metastasis. In the present report, we demonstrate real-time visualization of cell cycle kinetics of invading cancer cells in 3-dimensional (3D) Gelfoam® histoculture, which is in vivo-like. A fluorescence ubiquitination cell cycle indicator (FUCCI) whereby G₀/G₁ cells express a red fluorescent protein and S/G₂/M cells express a green fluorescent protein was used to determine the cell cycle position of invading and non-invading cells. With FUCCI 3D confocal imaging, we observed that cancer cells in G₀/G₁ phase in Gelfoam® histoculture migrated more rapidly and further than cancer cells in S/G₂/M phases. Cancer cells ceased migrating when they entered S/G₂/M phases and restarted migrating after cell division when the cells re-entered G₀/G₁. Migrating cancer cells also were resistant to cytotoxic chemotherapy, since they were preponderantly in G₀/G₁, where cytotoxic chemotherapy is not effective. The results of the present report suggest that novel therapy targeting G₀/G₁ cancer cells should be developed to prevent metastasis.     

NUCLEIC ACID CONTENT OF HeLa S3 CELLS DURING THE CELL CYCLE: VARIATIONS BETWEEN CYCLES

The process of continuous resynchronization with excess thymidine provides sufficient cell material for accurate chemical determination of DNA and RNA in HeLa S3 cells at hourly intervals during the cell cycle. Total DNA is constant during the non-S phase portion of the cell cycle but varies widely among cycles of synchronous growth. Total cellular RNA content increases linearly in the G₁ phase and accelerates to a higher linear rate of accumulation, which remains constant during most of the S and G₂ phases. The ratios of early and late cycle rates of RNA accumulation are not constant among cycles.