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.     

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.     

Cultured human keratinocytes: discrimination of different cell cycle compartments based upon measurement of nuclear RNA or total cellular RNA content

Abstract Correlated measurements of total cellular RNA and DNA of cultured human keratinocytes by flow cytometry, followed by multivariate analysis, discriminate three distinct subpopulations of cells differing in RNA content. The first subpopulation is comprised of small cells resembling basal cells of epidermis, with low RNA content and long (100–300 h) generation times. The second subpopulation consists of keratinocytes resembling cells in the spinous layer of epidermis, characterized by increased RNA content and shorter (35–40 h) generation times. The third subpopulation consists of the largest, keratinohyalin-containing cells which remain in G₁ and undergo terminal differentiation. In contrast to total cellular RNA, correlated measurements of DNA and nuclear RNA reveal that: (1) entrance of all cultured cells from G₁ into S phase occurs only after accumulation of the same, threshold amount of nuclear RNA; hence there is only a single population of S + G₂+ M-phase cells; (2) there are two distinct subpopulations in G₁, one with minimal nuclear RNA content and another with increased RNA. Stathmokinetic experiments indicate that the G₁-phase cells with low nuclear RNA have distinctly longer residence times in G₁ compared to cells with high nuclear RNA content. Thus, measurements of the total cellular RNA versus nuclear RNA content reveal kinetically distinct cell subpopulations. Whereas total cellular RNA content correlates more with differentiation, nuclear RNA content reflects primarily the kinetic properties of the cell.     

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.     

Evidence for Chromosomal Macronuclear Substructures in Tetrahymena*†

SYNOPSIS.
Under the growth conditions employed, the G₁ macronucleus of Tetrahymena pyriformis HSM contains 7.4 × 10^-12 g DNA, the G₂ micronucleus 0.42 × 10^-12 g. DNA content from the Tetrahymena thermophila macronucleus did not significantly differ from that of HSM, but the micronucleus contained about twice as much DNA as the micronucleus of the HSM cells. The T. thermophila macronucleus contained on average enough DNA for ˜ 35 haploid micronuclear copies. A new spreading technic allowed separation of macronuclear substructures from cells of late G₂ to early G₁. Photometric determination of DNA content of 345 individual structures suggested the existence of 5 different-sized macronuclear structures with a DNA content corresponding to 2, 4, 8, and 16 × the basic values. Comparison of the DNA content of these structures with (a) mitotic micronuclear chromosomes and (b) meiotic micronuclear chromosomes of T. thermophila cells suggests that the 5 basic values of macronuclear structures derive from structures of micronuclear chromosomes. The micronuclear chromosomes of T. pyriformis may be oligotenic. It is suggested that these results further our understanding of macronuclear organization.     

Evaluation of the effects of cryopreservation of isolated erythrocytes and leukocytes of largemouth bass by flow cytometry

We examined the effects of separation and freezing on fish leukocyte and erythrocyte morphology by light microscopy and on DNA content as measured by flow cytometry (FCM). Leukocytes and erythrocytes of largemouth bass Micropterus salmoides were isolated by density gradient centrifugation of whole blood, and frozen in liquid nitrogen in a buffer containing DMSO as a cryopreservative. The coefficient of variation (CV) of the G₀/G₁ peak of the cells was used to assess variation in nuclear DNA content within cell populations before and after separation and freezing treatments. In erythrocytes, the CV did not change significantly (P>0.05) when nuclei were isolated and stained without freezing or when erythrocytes were frozen prior to nuclear isolation and staining. In leukocytes, freezing and thawing prior to isolation and staining of nuclei significantly increased the CV (P<0.05), and produced hyperdiploid shoulders of the G₀/G₁ peak. However, the CV of leukocyte nuclei that were isolated and stained prior to freezing and the CV of non-frozen leukocyte nuclei did not differ (P>0.05). Microscopy showed that the freezing protocol had little effect on erythrocyte morphology, but caused irregular swelling in leukocytes. Freezing intact leukocytes also significantly (p<0.05) altered the apparent distribution of cells among the phases of the cell cycle as measured by FCM. The distributions of leukocyte nuclei that were isolated and stained prior to freezing were not different to non-frozen leukocytes. DNA measurements of nucleated blood cells are widely used in physiological, genetic and toxicological studies. Our results suggest that whole blood and erythrocytes for use in such studies can be frozen whole using a simple protocol, but leukocyte nuclei must be isolated and stained before freezing to avoid serious artifacts.     

Cytokinetic studies on the switch from glucose to uridine metabolism, and vice versa, of Ehrlich ascites tumour cells in vitro

Abstract. Glucose is normally required as the energy source and for the proliferation of neoplastic cells. For Ehrlich ascites tumour cells, kept under glucose-free culture conditions, this requirement was alleviated by uridine, indicating that the supply of ribose is obligatory for sustaining growth capacity.
In a 96-hr culture experiment with mouse-derived cells, the increase in cell number from cultures supplemented with 5 mM uridine was 50–70%, whilst lactate production was 5% that of controls. An increase in the number of multinucleate cells was observed from cell-smears; DNA histograms indicated the presence of cells with a DNA content higher than 4c and an increased portion of cells in G₂ phase. For precise determination of changes in cell cycle distribution on transfer of cells from glucose-supplemented to glucose-free conditions, the progression of phase-accumulated cells (by centrifugal elutriation) was monitored by DNA distribution analysis; G₂ cells continued the cycle at a rate comparable to controls but were delayed, in the following cycle, predominantly in S and G₂ phases. This was also observed with G₁ cells from a G₁-accumulated fraction in the first cycle.
The addition of glucose to cells kept for some hours in glucose-free, uridine-supplemented medium resulted in an immediate increase in mitotic index (amplification by the colcemid method).
The results are interpreted and support our concept that the delivery of compounds, necessary for normal growth, i.e. hexoses for glycoproteins and glycolipids, are limited as a consequence of the 'metabolic channelling' of pentose from uridine in Ehrlich ascites tumour cells. Therefore, the constantly lowered growth-rate in uridine-supplemented cells observed with long-term culture experiments could reflect an adaption of growth-cycle to these limitations.     

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.     

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.     

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.     

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.     

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.     

Flow cytometric correlation of the c-myc oncoprotein and cell cycle kinetics of HL60 leukaemia during induced maturation with cytosine arabinoside and dimethylsulphoxide

Abstract The c-myc oncogene codes for a DNA binding protein that functions in a cell cycle-related manner. A useful model for studying the relationship of c-myc expression with cell cycle kinetics is the HL60 cell line. HL60 cells constitutively express high levels of c-myc mRNA; however, the level can be down-regulated as the cells are induced to differentiate. We have developed a flow cytometric assay for correlating c-myc oncoprotein levels with DNA content. C-myc oncoprotein levels were additionally correlated with c-myc mRNA levels as determined by slot blot hybridization. Dimethylsulphoxide (DMSO) and cytosine arabinoside were used to induce granulocytic and monocytic maturation respectively. Treatment of HL60 cells with DMSO leads to an increase in the per cent of cells in G₁/G₀ and a decrease in mean c-myc mRNA and oncoprotein levels. The cells with G₁ DNA content show the greatest decrease in c-myc protein. ARA-c treatment of HL60 cells leads to a slowing and an accumulation of cells in S phase with a moderate decrease in mean mRNA and only a slight decrease in mean c-myc protein levels. These data support the hypothesis that c-myc is involved in the switch from G₁ to G₀.     

Evidence for altered cell-cycle traverse of the non-modal cells of the heteroploid MCa-11 line

Classic stem cell theory states that the growth of heteroploid cell populations is due to the proliferation of 'main stemline'cells with modal DNA content and chromosome number. Cells with non-modal DNA content and chromosome number are thought to be blocked and/or destroyed at mitosis. To test this, we studied two chromo-somally stable cell populations (mouse bone marrow and WCHE-5 cells) and one heteroploid, chromosomally diverse cell line (MCa-11). The heteroploid MCa-11 cells showed significant [³H]dT labelling for cells with DNA contents below the modal G_o/G₁ peak and above the modal G₂ peaks ( P <0.001). This was consistent with the presence of cells with the non-modal DNA content that were engaged in replicative DNA synthesis. A percentage labelled mitosis analysis showed that MCa-11 cells with non-modal DNA content and chromosome number were able to complete mitosis, although with prolonged pre-karyokinetic time. These results suggest that many non-modal cells present in heteroploid cell populations are capable of continued proliferation.     

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.     

Tetraploid cycle in ageing solid tumours

Abstract. When the mouse mammary adenocarcinoma 755 (Ca-755) reaches the plateau phase of growth, non-cycling cells with a G₂-DNA content can be observed. They may belong to the diploid cell cycle but they could also be blocked in G₀ or G₁ of a tetraploid cycle. This hypothesis was tested in three ways: (1) non-cycling G₂ nuclei were stained with a combination of Feulgen and naphthol yellow which revealed two populations, one with a low protein content and the other with a high protein content– the latter may represent nuclei ready to begin a new phase of DNA synthesis; (2) Feulgen staining and autoradiography were performed after tritiated thymidine had been administered to mice continuously: this showed that there were cells synthesizing DNA with a DNA index above 2; and (3) cells having 80 chromosomes, corresponding to the tetraploid cycle, were found almost exclusively in the plateau phase tumours.
On the other hand, the use of texture and DNA parameters of the Feulgen stained nuclei showed that they were concentrated in a diploid cycle for tumours in the exponential phase of growth and were divided between a diploid and tetraploid cycle for 'plateau' cells. Neither the cause for, nor the role played by, polyploid cells is known.     

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.     

CELL CYCLE PROPERTIES OF DIFFERENTIATING SPERMATOGONIA IN ADULT SPRAGUE-DAWLEY RATS

The duration of the mitotic cycle and of its components was analysed for each of the six successive generations of differentiating spermatogonia (A₁, A₂, A₃, A4, intermediate and B), using radioautographed whole mounts of seminiferous tubules from testes of adult Sprague-Dawley rats. Cell cycles were determined from two successive waves of per cent labeled metaphases obtained during the period of 81 hr after a single dose of ³H-thymidine. Except for the A₁ spermatogonia, all spermatogonial types (A₂ to B) had similar cell cycle durations of 41-42.5 hr and comparable pre-DNA synthesis phases (G₁) of 11-13 hr. Although the combined duration of DNA synthesis (S) and the post-synthesis phase (G₂) remained identical for all the cell types including A₁, there was a progressive lengthening of the S period at the expense of G₂ during the process of spermatogonial maturation. This change was most marked during the transition from A₁ to A₃ spermatogonia when the S period increased from 14 hr to 21 hr, and the G₂ phase shortened from 13 hr to 7.5 hr. This feature seems to be unique to germ cells and may be associated with an increasing amount of heterochromatin in the nucleus. Excluding the development of type A₁ cells, the entire process of spermatogonial maturation lasted for 208 hr. Combined data on cell cycle times indicated that every 313 hr or 13 days, a new sequence of spermatogonial differentiation was initiated by the A₁ cells. This was equivalent to the duration of one 'cycle' of the seminiferous epithelium as measured by other techniques.     

Induction of replicative senescence by 5-azacytidine: fundamental cell kinetic differences between human diploid fibroblasts and NIH-3T3 cells

Abstract. To analyse the putative role of methylation of cytosine residues in the nuclear DNA as a regulatory step during cellular ageing, we incubated ageing human amniotic fluid derived fibroblast-like cells and non-ageing NIH-3T3 cells with 5-azacytidine. BrdUrd/Hoechst and acridine orange (AO) flow cytometry was used to compare the effects of the base analogue on cell proliferation and cell differentiation. In NIH-3T3 cultures, 96 h exposures to 4 μM 5-azacytidine caused diminished cell proliferation due to cell arrest in the G₁ compartments of the second and third cell cycles of serum stimulated cells. The exit from the G₀/G₁ compartment was not affected. The 5-azacytidine induced cell kinetic disturbances were unstable in NIH-3T3 cultures, such that pre-treated cells reverted to normal cell cycle transit within 2–3 days after termination of treatment. In contrast, 5-azacytidine pre-treated amniotic fluid derived fibroblast-like cell cultures showed persistently elevated G₂ phase arrests and delayed G₀/G₁ phase exit kinetics, which explain the premature cessation of proliferation observed in these primary cultures. In both cell systems, 5-azacytidine exposed cultures showed elevated numbers of G₁ phase cells with increased RNA content as revealed by AO flow cytometry. Again, this effect was reversible in NIH-3T3 cells but not in amniotic fluid derived fibroblast-like cells. These contrasting responses to 5-azacytidine are likely to reflect intrinsic differences in methylation patterns or de novo methylase activity between ageing cell strains and non-ageing cell lines.