EFFECTS OF PHYTOCHROME AND BLUE-NEAR ULTRAVIOLET LIGHT-ABSORBING PIGMENT ON DURATION OF COMPONENT PHASES OF THE CELL CYCLE IN ADIANTUM GAMETOPHYTES

Single-celled protonemata of the fern Adiantum capillus-veneris, kept under continuous red light, grew with a very low rate of cell division, and the cell cycle was arrested in the early G₁ phase. Cell division was induced by transferring the protonemata to the dark after various light treatments, and the duration of component phases in the cell cycle was determined by a continuous-labelling technique with 3H-thymidine. Blue light irradiation greatly reduced the duration of the G₁ phase but did not affect that of other phases. The greater the fluence of blue light, the shorter was the duration of G₁ phase was observed. In contrast, a brief exposure of red-light-grown protonemata to far-red light given immediately before the dark incubation showed no effect on the duration of G₁ S and M phases but significantly extended that of the G₂ phase. The effect of far-red light on the G₂ phase was reversed by red light, and the effects of red and far-red light were repeatedly reversible. The progression in the M phase was shown by means of a time-lapse video system to be not at all influenced by any pre-irradiation described above.     

The effects of interleukin 4 on the cell cycle of a human B-cell lymphoma line

Abstract. Exposure of Farage, a human B-cell lymphoma line, to IL-4 for 3–11 days led to inhibition of tritiated thymidine ([³H]dT) uptake by the cells. Study of the incorporation of 5-bromodeoxyuridine by Farage cells showed that IL-4 reduced significantly the number of cells in the S phase of the cell cycle and increased the proportion of cells in the G₁ phase. Limiting dilution analysis of proliferation demonstrated that IL-4 decreased the frequency of clone-forming cells by 40%. IL-4 did not reduce the viability of Farage cells. On the contrary, IL-4 diminished the spontaneous death of Farage cells in culture, as determined by pulse chase analysis of cells which were labelled with [³H]dT. Moreover, the pre-treatment of Farage cells with IL-4 prevented their death induced by exposure to a high dose of staurosporine. IL-4 abrogated the staurosporine-induced arrest of cells in the G₂+ M phase and replaced it by accumulation of cells in the G₁ phase. IL-4 protected Farage cells from the radioactive suicide caused by the uptake of [³H]dT by dividing cells. The cytokine failed to prevent the damage to Farage cells exerted by mitomycin C, which affected cellular DNA regardless of the phase of the cell cycle. The data obtained showed that IL-4 inhibited the division of B cells by arresting their progression through the early stages of the cell cycle. This inhibition of the cell efflux from G₁ phase plays an important role in the protection against cell death during further stages of the cell cycle.     

Expression of G1 and G2 cyclins measured in individual cells by multiparameter flow cytometry: a new tool in the analysis of the cell cycle

Abstract. Multivariate analysis of the expression of cyclin proteins and DNA content has opened new possibilities for the study of the cell cycle. By virtue of their cell cycle phase specificity, the expression of cyclins may serve, in addition to DNA content, as another marker of a cell's position in the cycle, and provide information about the proliferative potential of cell populations. Several applications of the methodology based on bivariate analysis of DNA content v . expression of B, E and D type cyclins are reviewed: 1 expression of cyclins by individual cells during their progression through the cycle can be studied, using exponentially growing cells without the necessity of cell synchronization or other perturbations of the cycle; 2 cells having the same DNA content but residing in different phases of the cycle (e.g. G₂ diploid v. G₁ tetraploid) can be distinguished; 3 cell transition from G₀ to G₁ and progression through G₁ (e.g. mitogen stimulated lymphocytes) can be assayed; 4 the population of proliferating cells can be distinguished from noncycling cells based on dual cell labelling with a G₁ and G₂ cyclin antibody; 5 cyclin restriction points can serve as additional cell cycle landmarks to map the point of action of antitumour drugs; 6 unscheduled expression of cyclins (e.g. the presence of cyclin B1 during G₁ and S) can be detected in several tumour transformed cell lines, possibly indicating disregulation of the machmery of cell cycle progression. The last finding 6 is of special importance, because such disregulation may be of prognostic consequence in human tumours.     

Heterogeneity in the mouse epidermal cell cycle analysed by computer simulations

Abstract. Different sets of cell kinetic data obtained over many years from hairless mouse epidermis have been simulated by a mathematical model including circadian variations. Simulating several independent sets of data with the same mathematical model strengthens the validity of the results obtained. The data simulated in this investigation were all obtained with the experimental system in a state of natural synchrony. The data include cell cycle phase distributions measured by DNA flow cytometry of isolated epidermal basal cells, fractions of tritiated thymidine ([³H]TdR) labelled cells within the cell cycle phases measured by cell sorting at intervals after [³H]TdR pulse labelling, bivariate bromodeoxyuridine (BrdUrd)/DNA data from epidermal basal cells isolated at intervals after pulse labelling with BrdUrd, mitotic rate and per cent labelled mitosis (PLM) data from histologic sections. The following main new findings were made from the simulations: the second PLM peak observed at about 35 h after pulse labelling is hardly influenced by circadian variations; the peak is mainly determined by persisting synchrony of a rapidly cycling population with a G₁-duration (T_G1) of 20 h to 30 h; and there is a highly significant population of slowly cycling G₁-cells (G_1σ). However, no significant circadian variations were found in the number of these cells.     

Long duration of mitosis and consequences for the cell cycle concept, as seen in the isthmal cells of the mouse pyloric antrum. II. Duration of mitotic phases and cycle stages, and their relation to one another

Abstract. The kinetics of isthmal cells in mouse antrum were examined in three ways: (a) the duration of cell cycle and DNA-synthesizing (S) stage was measured by the 'fraction of labelled mitoses' method; (b) the duration of interphase and mitotic phases was determined from how frequently they occurred; and (c) mice were killed at various intervals after an intravenous injection of ³H-thymidine to time the acquisition of label by the various phases of mitosis.
The duration of the isthmal cell cycle was found to be 13.8 hr and that of the DNA-synthesizing (S) stage, 5.8 h. Estimates for the duration of the G₁ and G₂ stages were 6.8 and 1.0 hr, respectively.
From the frequency of mitotic phases, defined as indicated in the preceding article (El-Alfy & Leblond, 1987) and corrected for the probability of their occurence, it was estimated that prophase lasted 4.8 hr; metaphase, 0.2 hr; anaphase, 0.06 hr and telophase, 3.3 hr, while the interphase lasted 5.4 hr. In accordance with this, the duration of the whole mitotic process was 8.4 hr.
Ten minutes after an intravenous injection of ³H-thymidine, 38% of labelled isthmal cells were in interphase and 62% in early or mid prophase, while cells in late prophase and other mitotic phases were unlabelled. After 60 min, label was in late prophase, after 120 min, in mid telophase and after 180 min, in late telophase.
We conclude that there is overlap between some mitotic phases and cycle stages. Thus, while nuclei are at interphase during the early third of S, they are in prophase during the late two-thirds as well as during G₂. Also, nuclei are in telophase during the early half of G₁ but at interphase during the late half. Differences in nuclear diameter show that subdivision of both S and G₁ into early and late periods is practical.     

Influence of Growth Hormone and Thyroxine On Cell Kinetics In the Proximal Tibial Growth Plate of Snell Dwarf Mice

Abstract. In Snell dwarf mice, the influence of short-term treatment with human growth hormone (hGH) or thyroxine on the proliferative and sulphation activity of the proximal tibial growth plate was studied. By autoradiographic methods, the [³H]methylthymidine incorporation after a single injection was measured, after 2 hr incorporation time. the labelling index was calculated and the number of labelled mitoses was counted. In addition, the distribution of the labelled nuclei over the proliferating and degenerating zones was determined by continuous labelling for 25 and 73 hr.
In untreated dwarf mice after [³H]-methylthymidine administration, the number of labelled nuclei in the growth plate is low. Labelling occurs, as expected, mainly in the cells of the proliferative zones. the number of labelled nuclei in control dwarf mice was similar after 25 and 73 hr continuous labelling. This suggests that many cells are in a resting G_o or prolonged G₁ phase. Both hGH and T₄ treatment induce a significant increase of the number of labelled nuclei per growth plate and of the number of mitoses. Since hormonal treatment induces a small number of mitoses after 2 hr incorporation of the label, the minimal G₂ phase of the cell cycle is less than 2 hr. In addition, treatment with hGH and T₄ stimulates chondrocytes in the zone of proliferative and hypertrophic cells to actively incorporate [³⁵S]-sulphate.     

ON THE RESTING STAGES OF THE JB-1 ASCITES TUMOUR

Cytophotometric determination of single-cell DNA after repeated ³H-thymidine labelling of the JB-1 ascites tumour in the plateau phase of growth showed a massive accumulation of unlabelled cells with both G₁ and G₂ content. Autoradiography combined with cytophotometry or colcemid block demonstrated that some of these unlabelled cells were rapidly triggered into the cell cycle when plateau tumours were transferred to new hosts. This indicated that tumour cells may be held up in non-cycling stages corresponding to both the G₁ and the G₂ phase of the cell cycle.     

EFFECT OF CENTRIFUGATION ON THE DEVELOPMENT AND TIMING OF PREMITOTIC POSITIONING OF THE NUCLEUS IN ADIANTUM PROTONEMATA

When single-celled protonemata of Adiantum capillus-veneris L. were centrifuged immediately before transferring to darkness from continuous irradiation with red light, their nuclei were displaced basipetally. Both filamentous and branched protonemata were obtained. The stronger the centrifugal acceleration, the more frequently the branched protonemata were induced.
The effect of centrifugation at 1,300 x g for 15 min on nuclear displacement was different at different stages of the cell cycle. In early G₁ phase, the nucleus was easily displaced by centrifugation, but quickly returned to the original position after centrifugation. In late G₁ phase, the nucleus was displaced, but after centrifugation it never came back to the original position. In late G₂ and M phases, the nucleus was no longer displaced by the centrifugation. Premitotic positioning of the nucleus in cytokinesis took place about 5 hr before cell plate formation in all centrifugal treatments described above.     

New methods for calculating kinetic properties of cells in vitro using pulse labelling with bromodeoxyuridine

Abstract. The transit times of Chinese hamster ovary cells through the phases of their cell cycle were measured using dual parameter flow cytometry to measure DNA content and the presence of monoclonal antibodies to bromodeoxyuridine. Up to four separate populations can be accurately measured: unlabelled cells in G₂+ M; labelled cells that have not yet divided; labelled cells that have already divided; and the unlabelled cells that were originally in G₁ plus the cells that were originally in G₂+ M and have since divided. The fractions of cells in these populations can be easily followed in time and the usual kinetic properties can be estimated from these fractions, or combinations thereof, including the times through G₁, S, G₂+ M and the cycle time. We present equations for analysing this type of data and comment on which equations are most appropriate for measuring specific kinetic properties of the cells.     

Growth pattern of the human promyelocytic leukaemia cell line HL60

Abstract. In order to characterize the growth pattern of the human promyelocytic leukaemia cell line HL60, its kinetic parameters were studied. The doubling time was calculated from serial cell counts, the duration of the various cell cycle phases from the analysis of the labelled mitoses curve, and quiescent population from continuous labelling experiments. Proliferation in culture was exponential up to a saturation density of about 3.0 × 10⁶ cells/ml, with a doubling time of 34.0 hr. The cell cycle duration was 24.3 ± 4.1 hr (SD), and that of the cell cycle phases was: G₁, 3.8 ± 2.2 hr; S, 15.1 ± 3 hr; and G₂, 5.4 ± 1.2 hr. The growth fraction was 0.85, and cell loss was restricted to the quiescent cells. The HL60 cell line, with fully characterized kinetics, provides a useful tool for the in vitro study of substances which may affect human leukaemic myelopoietic proliferation.     

Synthesis of protein and mRNA is necessary for transition of suspension-cultured Catharanthus roseus cells from the G1 to the S phase of the cell cycle

The effects of inhibition of the synthesis of protein, mRNA or rRNA on the progression of the cell cycle have been analyzed in cultures of Catharanthus roseus in which cells were induced to divide in synchrony by the double phosphate starvation method. The partial inhibition of protein synthesis at the G₁ phase by anisoniycio or cycloheximide caused the arrest of cells in the G₁ phase or delayed the entry of cells into the S phase. When protein synthesis was partially inhibited at the S phase, cell division occurred to about the same extent as in the control. When asynchronously dividing cells were treated with cycloheximide, cells accumulated in the G₁ phase, as shown by flow-cytometric analysis. The partial inhibition of mRNA synthesis by α-amanitin at the G₁ phase caused the arrest of cells in the G₁ phase, although partial inhibition of mRNA synthesis at the S phase had little effect on cell division. In the case of inhibition of synthesis of rRNA by actinomycin D at the G₁ phase, initiation of DNA synthesis was observed, but no subsequent DNA synthesis or the division of cells occurred. However, the addition of actinomycin D during the S phase had no effect on cell division. These results suggest that specific protein(s), required for the progression of the cell cycle, are synthesized in the G₁ phase, and that the mRNA(s) that encode these proteins are also synthesized at the G₁ phase.     

Effect of separase depletion on ionizing radiation-induced cell cycle checkpoints and survival in human lung cancer cell lines

Abstract.   Objectives : This study is to evaluate the effect of separase depletion on cell cycle progression of irradiated and non-irradiated cells through the G₂/M phases and consecutive cell survival. Materials and methods : Separase was depleted with siRNA in two human non-small cell lung carcinoma (NSCLC) cell lines. Cell cycle progression, mitotic fraction, DNA repair, apoptotic and clonogenic cell death were determined. Results : By depletion of endogenous separase with siRNA in NSCLCs, we showed that separase affects progression through the G₂ phase. In non-irradiated exponentially growing cells, separase depletion led to an increased G₂ accumulation from 17.2% to 29.1% in H460 and from 15.7% to 30.9% in A549 cells and a decrease in mitotic cells. Depletion of separase significantly ( P <  0.01) increased the fraction of radiation-induced G₂ arrested cells 30–56 h after irradiation and led to decrease in the mitotic fraction. This was associated with increased double-strand break repair as measured by γ-H2AX foci kinetics in H460 cells and to a lesser extent in A549 cells. In addition, a decrease in the expression of mitotic linked cell death after irradiation was found. Conclusions : These results indicate that separase has additional targets involved in regulation of G₂ to M progression after DNA damage. Prolonged G₂ phase arrest in the absence of separase has consequences on repair of damaged DNA and cell death.     

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

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

Cell cycle related [3H]thymidine uptake and its significance for the incorporation into DNA

Abstract. Cellular uptake of [³H]thymidine ([³H]TdR) and incorporation into DNA of Ehrlich ascites tumour cells were studied in relation to the cell cycle by measuring the activity in the acid-soluble and insoluble parts of the cell material. Cells were synchronized at various stages of the cell cycle using centrifugal elutriation. The degree of synchrony of the various cell fractions was measured by flow-cytofluorometric DNA analysis. From the cellular uptake, the TdR triphosphate (dTTP) concentration of a mean cell in an unseparated cell population was calculated to be 20 × 10^-18 mol/cell. The pool activity of G₁ cells was unmeasurable but rose to maximum values at the border of the G₁-S phase. It decreased again during G₂. The [³H]TdR incorporation into DNA was low during early S phase, reached a maximum value at two-thirds of the S phase and decreased again during late S phase. These changes in DNA synthesis were not due to changes in the dTTP pool being a limiting factor. During maximum DNA synthesis, 10%× min^-1 of the dTTP pool was utilized, at which time the pool size also decreased by about 30%. Changes in pool size during the cell cycle have to be taken into account when the results of incorporation of radioactive TdR into DNA are discussed.     

Normal G1/S transition and prolonged S phase within one cell cycle after seeding cells in the presence of an ornithine decarboxylase inhibitor

Abstract. We have previously found that DNA replication was affected within one cell cycle after seeding Chinese hamster ovary (CHO) cells in the presence of the polyamine biosynthesis inhibitor 2-difluoromethylornithine (DFMO). We could, however, not rule out if this was due to an effect on the G₁/S transition and/or on DNA synthesis elongation. In the present paper, we use a bromodeoxyuridine-flow cytometric method to more specifically study the G₁/S transition, the S phase length, and the progression of cells from S phase through G₂+ M and into G₁, after seeding plateau phase CHO cells at low density in the absence or presence of 5 mM DFMO. We report here that DFMO-induced polyamine depletion increased the length of the S phase within one cell cycle after seeding of CHO cells in the presence of the inhibitor. No effect on the G₁/S transition was observed until 2 days after seeding, suggesting that a DFMO-induced lengthening of the G₁ phase occurred later than the effect on S phase progression. These results imply that the G₂+ M phase was not prolonged until 2 days after seeding CHO cells in the presence of DFMO.     

Age-related changes in the cell kinetics of rat foot epidermis

Abstract. The durations of the cell cycle and its component phases have been determined for the basal layer of the epidermis of the skin from the upper surface of the hind foot of the rat using single pulse [³H]-thymidine labelling and the percent labelled mitosis (PLM) technique. Rats of three age groups were used, namely 7, 14 and 52 weeks. The duration of DNA synthesis (T_s) and the G₂ plus M phase (Tg₂± m) were comparable in 7-week and 52-week-old rats ( P > 0–1). The major difference between 7-week and 52-week-old rats was in the duration of the G₁ phase (Tg₁). In 7-week-old rats Tg₁ was 15.0 ± 0.8 h and in 52-week-old rats Tg₁ was 31.2 ± 3.5 h. A consequence of this variation was that the overall duration of the cell cycle was longer in 52-week-old rats (53.9 ± 5.3 h) than in 7-week-old rats (30.1 ± 1.3 h).
Difficulties were found in fitting a simple curve to the PLM data for 14-week-old rats. This suggests that the proliferative cell population of the epidermis of rats of this age group may be heterogeneous. A satisfactory fit to the data was obtained using a computer model which assumed that the proliferative population of the epidermis of 14-week-old rats was a mixture of cells with cell cycle parameters the same as those of the 7-week and the 52-week-old rats. These two sub-populations of relatively slowly and rapidly proliferating cells were present in the ratio of 2:1.     

Resumption of cellular activity induced by cytokinin and gibberellin treatments in tomato flowers targeted for abortion in unfavorable light conditions

Mitotic activity and nuclear DNA synthesis in tomato ( Lycopersicon esculentum Mill., cv. King plus) flowers targeted for abortion under unfavorable light conditions are completely stopped 6 days after macroscopic appearance of the inflorescence. Ovular cells are arrested at the G₁ (80%) and G₂ (20%) stages of the cell cycle. Exogenous applications of a mixture of N⁶-benzyladenine (BA) and gibberellins A₄₊₇ (GA) directly on the inflorescence may prevent its failure. Nuclear DNA synthesis and mitoses resume in ovules of the flower 16 to 20 h after the BA+GA treatment. When applied alone, BA and GA are able to mimic the effect of the mixture upon the progression of ovular cells through their cycle. Sporogenesis processes are also set in motion by the exogenous plant growth regulators. The mechanism of action of cytokinins and gibberellins in the control of floral development is discussed.     

Mammalian cells are not synchronized in G1-phase by starvation or inhibition: considerations of the fundamental concept of G1-phase synchronization

Synchronization of mammalian cells by starvation-refeeding or by inhibition-release are among the most commonly used techniques for division cycle analysis. An alternative analysis—in the form of a Gedanken or thought experiment—is presented, casting doubt on the utility of this synchronization method. Arresting cell growth produces a culture where all cells contain a G₁ amount of DNA. However, these cells are not arrested at a particular point in the G₁-phase. Analysis of 'G₁ arrested cells' suggests that, upon resumption of growth, the cells are not synchronized.     

Monocytic differentiation of HL60 cells induced by potent analogs of vitamin D3 precedes the G1/G0 phase cell cycle block

Abstract. Differentiation of mammalian cells is accompanied by reduced rates of proliferation and an exit from the cell cycle. Human leukemic cells HL60 present a widely used model of neoplastic cell differentiation, and acquire the monocytic phenotype when exposed to analogs of vitamin D₃ (VD₃). The maturation process is accompanied by two blocks in the cell cycle: an arrest in the G₁/G₀ phase, and a recently described G₂+ M block. In this study we have analyzed the traverse of the cell cycle phases of the well-differentiating HL60-G cells exposed to one of ten analogs of VD₃, and compared the cell cycle effects of each compound with its potency as a differentiation-inducing agent. We found that in general there was a good correlation between the effects of these compounds on the cell cycle and on differentiation, but the best cell cycle predictor of differentiation potency was the extent of accumulation of the cells in the G₂ compartment. All analogs induced a marked decrease in the mitotic index, and polynucleation of HL60 cells was produced, especially by compounds which were effective as inducers of differentiation. Time course studies showed that induction of differentiation was accompanied by a transient increase of the proportion of cells in the G₂+ M compartment, but preceded the G₁ to S, and the G₂ compartment blocks. These studies indicate that complex changes in the cell cycle traverse accompany, but do not precede, the acquisition of the monocytic phenotype by HL60 cells.