RESPONSE OF MOUSE BONE MARROW COLONY FORMING UNITS IN DIFFERENT STAGES OF THE CELL CYCLE TO IN VITRO INCUBATION WITH MITOMYCIN-C

A short-term in vitro method was employed to study the Mitomycin-C sensitivity of normal mouse bone marrow CFU without triggering the G₀-phase cells into the proliferative cycle. Comparison was made of the toxicities of the drug against cells in different phases of the cell cycle including G₀. Mitomycin-c killed CFU both in and out of the S-phase. No significant difference could be found between its toxicities against normal and proliferating CFU; along the exponential part of the survival curve 1·6 μg/ml concentration of the drug reduced survival to 10%. Although in the normal bone marrow only a few CFU are in the S-phase and are killed by the agent, presence of the sensitive G₀ cells produce a significant amount of non-S-phase mortality. Among the proliferating CFU population the non-S-phase lethality is less due to the absence of G₀ cells. About 75% of the S-phase cells are killed after incubation with 1 μg/ml drug; outside the S-phase, the lethality is about 40–50%. The studies indicate that the G₀ cells which are situated near the G₁-S boundary are almost as sensitive to the drug as other non-S-phase cells like G₁ cells. The clinical significance of the findings is discussed.     

EFFECTS OF ACTINOMYCIN D AND PUROMYCIN ON THE CELL PROGRESS FROM M TO G1 AND S STAGES IN CULTURED MOUSE LEUKEMIA L5178Y CELLS

Actinomycin D (0.5 μg/ml) did not prevent M stage cells from entering G₁ stage, but blocked their progress from G₁ to S stage. The position of the block was approximately 1.4 hr before S stage or just after the beginning of G₁ stage. Actinomycin D in this concentration also significantly depressed uridine-³H uptake into G₁ stage cells, but did not suppress leucine-³H uptake by M and G₁ cells. This suggests that some proteins may be synthesized in M and G₁ stage cells by messenger RNA left over from the previous cell cycle. However, entry of G₁ cells into S stage would require synthesis of new messenger RNA near the beginning of G₁ stage. Puromycin (10 μg/ml) did not prevent M cells from entering G₁ stage, but blocked their progress from G₁ to S stage. The site of blockage was about 0.7 hr before S stage or in the first two-third of G₁ stage. This might be the site where the cells synthesize new G₁ proteins necessary for entry to S stage.
Comparison of sensitivities of G₁ and G₂ stages to the two antibiotics reveals that the puromycin sensitivity of G₁ cells was similar to that of G₂ cells, but the actinomycin D sensitivity of G₁ was greater than that of G₂ cells.     

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.     

Method For Probing Cells In Radiation-Induced G2Arrest: Demonstration of Potentially Lethal Damage Repair

Abstract. Chinese hamster ovary cells were arrested in the G₂ phase of the cell cycle by X-irradiation. When subsequently treated with 5 mM caffeine the arrested population progressed into mitosis as a synchronous cohort where it was harvested by mitotic cell selection. This procedure provides a means to isolate cell populations treated in G₂, for the investigation of G₂ arrest. Comparisons were made of the number of cells retrieved from G₂ arrest with the number suffering arrest, as determined by flow cytometry and by matrix algebraic simulations of irradiated cell progression. the retrieved population was not significantly less than expected for doses up to 3.5 Gy, indicating that the retrieval process does not favour the isolation of any population subset below this dose. Cell populations retrieved from arrest at varying intervals (0-3 h) after irradiation (0-3.5 Gy) showed an increase in survival with increase in interval, consistent with repair of potentially lethal damage. the repair curves (surviving fraction us time) were each described by a single exponential. G₂ cells that were brought to mitosis without a period of arrest exhibited the same radiation response as cells irradiated in mitosis.     

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.     

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.     

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 SUBUNIT STRUCTURE OF MAMMALIAN ACETYLCHOLINESTERASE: CATALYTIC SUBUNITS, DISSOCIATING EFFECT OF PROTEOLYSIS AND DISULPHIDE REDUCTION ON THE POLYMERIC FORMS

Abstract— An analysis of the [³H]DFP-labelled catalytic subunits of mammalian (bovine SCG) acetylcholinesterase (AChE, EC 3.1.1.7.) indicates a monomer molecular weight of 75,000. This is equivalent to the mass previously determined for the smallest active form and demonstrates that the globular, or G forms, are respectively monomeric (G₁ form, 4S), dimeric (G₂ form, 6.5S) and tetrameric (G₄ form, 10S). In the tetrameric G₄ form the catalytic chains are associated in dimers, by disulphide bonds.
The effect of reduction and proteolysis has shown that the dimeric form (G₂ form, 6.5S) is readily reduced into G₁, while the tetramer G₄ is very stable, being only dissociated by a combination of reduction and proteolysis by high concentration of trypsin. The asymmetric forms A₁₂ (16S), A₈ (13S) and A₄ (9S) are not sensitive to reduction, but are readily dissociated by low concentrations of trypsin, into each other, progressively liberating isolated tetramers. We obtained essentially identical results with AChE preparations from rat brain or superior cervical ganglion. These observations support a general model for the quaternary structure of acetylcholinesterase molecular forms.     

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.     

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.     

cAMP IN THE CELL CYCLE OF THE DINOFLAGELLATE CRYPTHECODINIUM COHNII (DINOPHYTA)

The second messenger cAMP is a key regulator of growth in many cells. Previous studies showed that cAMP could reverse the growth inhibition of indoleamines in the dinoflagellate Crypthecodinium cohnii Biecheler. In the present study, we measured the level of intracellular cAMP during the cell cycle of C. cohnii . cAMP peaked during the G₁ phase and decreased to a minimum during S phase. Similarly, cAMP-dependent protein kinase activities peaked at both G₁ and G₂+M phases of the cell cycle, decreasing to a minimum at S phase. Addition of N6, O₂'-dibutyryl (Bt₂)-cAMP directly stimulated the growth of C. cohnii . Flow cytometric analysis of synchronized C. cohnii cells suggested that 1 mM cAMP shortened the cell cycle, probably at the exit from mitosis. The size of Bt₂-cAMP treated cells at G₁ was also larger than the control cells. The present study demonstrated a regulatory role of cAMP in the cell cycle progression in dinoflagellates.     

Cyclin B1 expression in response to abrogation of the radiation-induced G2/M block in HeLa cells

The G₂ block is a major response of cells to DNA damage and seem to be induced independently of p53 status. It is thought that the G₂ block has a protective function and allows cells to repair their DNA. The molecular events involved in the formation of the G₂ block therefore are of great interest. We have used pentoxifylline, a potent G₂ delay abrogator, to study the expression of an essential component of the mitosis promoting complex (MPF), cyclin B1. Cyclin B1/G₂ ratios are used to show that irradiation induces a decrease in cyclin B1 expression and that pentoxifylline restores cyclin B1 expression to control level. This confirms that suppression of cyclin B1 plays a role in the formation of the G₂ cell cycle delay, and that elevating cyclin B1 expression is part of the mechanism of action of pentoxifylline on G₂ blocked cells.     

A comparison of different methods to determine cell proliferation by flow cytometry

Abstract. The proliferation of human melanoma cells (MeWo) in vitro was studied with a number of different techniques. In particular, we compared the expression of PCNA and the Ki-67 antigen on the one hand with BrdU pulse and continuous labelling on the other. Two-dimensional flow cytometry (with DNA content as a second parameter) was employed to discriminate between cycling and non-cycling cells as well as cells in the G₁, S and G₂ phases of the cycle. Cell cultures in different stages of growth were analyzed. We found that the percentage of anti-PCNA and Ki-67 positive cells agreed very well with the BrdU pulse and continuous labelling index, respectively. Our data further support the assumption that under certain conditions PCNA is a marker of S-phase cells, whereas Ki-67 can be used to quantify the growth fraction. Possible pitfalls of the techniques are discussed.     

Unbudded G2 as well as Gj arrest in the stationary phase of the basidiomycetous yeast Cryptococcus neoformans

Abstract Stationary-phase cells of Cryptococcus neoformans displayed two morphological characteristics: virtually all the cells were unbudded even in the early stationary phase and even when grown in rich media, and average cell size increased from that of exponential-phase cells. DNA contents for small and large stationary-phase cells were determined by quantitative fluorescence microscopy after DNA staining with propidium iodide or DAPI. Small cells contained G, DNA, whereas large unbudded cells had either a G₂ or G₁ DNA content, indicating that Cr. neoformans can enter into the stationary phase from either the G₁ or G₂ period.     

CELL CYCLE COMPARTMENT ANALYSIS OF CHINESE HAMSTER CELLS IN STATIONARY PHASE CULTURES

The distribution of Chinese hamster cells with respect to the compartments of the cell generation cycle was studied in cultures in the stationary phase of growth in two different media. A measure of the state of depletion of the nutrient medium was formulated by defining a quantity termed the nutritive capacity of the medium. This quantity was used to verify that the cessation of cell proliferation is due to nutrient deficiencies and not to density dependent growth inhibition. Cell cultures in stationary phase were diluted into fresh medium and as growth resumed, mitotic index, cumulative mitotic index, label index and viability were measured as a function of time. The distribution of cells with respect to compartments of the cell generation cycle in stationary phase populations was reconstructed from these data. Stationary phase populations of Chinese hamster cells that retained the capacity for renewed growth when diluted into fresh medium were found to be arrested in the G₁ and G₂ portions of the cycle; the relative proportion of these cells in G₁ increased with time in the stationary phase, but the sequence differs in the two media. In early stationary phase, in the less rich medium, more cells are in G₂ than in G₁. Also in this medium a fraction of the population was observed to be synthesizing DNA during stationary phase, but this fraction was not stimulated to renewed growth by dilution into fresh medium.     

Is there an accumulation of cells during G2 in some acute lymphoblastic leukaemias?

Abstract. In some cases of acute lymphoblastic leukaemia (ALL) the percentage of cells in G₂+ M is higher than anticipated when compared with the percentage in S phase. This increase in G₂+ M, as detected by flow cytometry measurement of DNA content, may be due to an accumulation of cells, either in G ₂ or during the end of S phase; it may also be related to the existence of small tetraploid clones generally ignored by cytogeneticists. In order to identify possible subpopulations of cells with a DNA index ≥ 2-0, we have compared the results of a cytogenetic analysis to the G₂+ M values. We have also studied the distribution of S phase cells in 24 cases of ALL by incorporating 5-bromodeoxyuridine, labelling the cells by indirect immunofluorescence, and analysing them by flow cytometry after propidium iodide staining. The distribution of cells during S phase was quantified: no accumulation of cells was ever observed at the end of S phase. The question of the existence of small tetraploid clones, G₂ arrested cells or cells with a G₂ elongation remains open. However, we feel that it is more probable that, in this pathology, an elongation of the duration of G₂ occurs.     

Cell cycle phase dependent emergence of thymidylate synthase studied by monoclonal antibody (M-TS-4)

Abstract A method of identifying thymidylate synthase (TS) at the cellular level was developed using anti-TS monoclonal antibody (M-TS-4), a monoclonal antibody created against purified TS from a HeLa cell line. In HeLa cells and four human glioma cell lines (U-251, U-87, 343-MGA, and SF-188), TS was identified primarily in the cytoplasm. Autoradiographic and flow cytometric studies showed that TS appeared mainly in the G₁ phase and subsided early in the S phase; thus, the G₁ phase can be divided into TS-positive and -negative fractions. Nuclear TS was not demonstrated unequivocally with M-TS-4, and the relationship between nuclear TS and DNA synthesis could not be determined. Although the percentage of TS-positive cells was larger than the S-phase fraction measured by autoradiography after a pulse of tritiated thymidine or by the immunoperoxidase method using BUdR, the ratios were within a similar range (1.2–1.4) in all cell lines studied. Therefore, the S-phase fraction can be estimated indirectly from the percentage of TS-positive cells measured by M-TS-4. Because the emergence of TS detected by our method is cell cycle dependent, M-TS-4 may be useful for biochemical studies of TS and for cytokinetic analysis.     

Flow cytometric analysis of the recruitment of G0 cells in human epidermis in vivo following tape stripping

Abstract. Tape stripping of human skin elicits a proliferative response of a synchronously-dividing group of cells. The progress of this cohort of cells has been monitored using two windows in the cell cycle, one located in mid-S phase and the other centred around G₂+ M. The cellular DNA is measured with flow cytometry, the windows are defined by two ranges in the DNA histogram.
The cohort can be described as the recruitment of cells from a pre-existing G₀ compartment which consists of 76% of all proliferative cells. The duration of the S phase is calculated to be 10.2 hr and G₂+ M phase 5.1 hr. The cell cycle time of 39 hr for normal human keratinocytes derived from these figures is in line with recent values obtained by different techniques.     

Protein Synthesis during Photocontrolled Progression of the Cell Cycle in Single-celled Protonemata of the Fern Adiantum Capillus-veneris

Protein synthesis during photoinduced, synchronous progression of the cell cycle in single-celled protonemata of the fern Adiantum capillus-veneris was studied by tracer techniques. Nuclei of the protonemata were labelled with ³H-thymidine during spore germination so that the amount of ³H incorporated into the TCA-insoluble fraction of the cells could be used as a measure of the cell number in each sample. The rate of the incorporation of ¹⁴C-amino acids into TCA-insoluble materials was not significantly varied at different stages of the cell cycle or by treatment with blue light. Extracts of cells labelled with ³⁵S-methionine at various times after the transfer from red light condition (G₀) to darkness (G₁ to S) were analyzed by two-dimensional gel electrophoresis. At least 3 of about 200 spots showed significant changes in intensity on fluorograms. Spot A (molecular weight 20,000, isoelectric point 6.3) was detectable only in early G₁, whereas spot B (molecular weight 19,500, isoelectric point 6.3) was found only in the late G₁ and S phases. When the cells were exposed to blue light before the dark incubation, the times of disappearance of spot A and appearance of spot B were advanced depending upon the progression of the cell cycle but not upon the clock time.