Different sensitivity of chromatin to acid denaturation in quiescent and cycling cells as revealed by flow cytometry.

The properties of DNA in situ as reflected by its staining with acridine orange are different in quiescent nonstimulated lymphocytes as compared with interphase lymphocytes that have entered the cell cycle after stimulation by mitogens. The difference is seen after cell treatment with buffers at pH 1.5 (1.3-1.9 range) followed by staining with acridine orange at pH 2.6 (2.3-2.9). Under these conditions the red metachromatic fluorescence of the acridine orange-DNA complex is higher in quiescent cells than in the cycling lymphocytes while the orthochromatic green fluorescence is higher in the cycling, interphase cells. The results suggest that DNA in condensed chromatin of quiescent lymphocytes (as in metaphase chromosomes) is more sensitive to acid-denaturation than DNA in dispersed chromatin of the cycling interphase cells. The phenomenon is used for flow cytometric differentiation between G0 and G1 cells and between G2 and M cells. In contrast to normal lymphocytes the method applied to neoplastic cells indicates the presence of cell subpopulations with condensed chromatin but with DNA content characteristic not only of G1 but also of S and G2 cells. The possibility that these cells represent quiescent (resting) subpopulations, arrested in G1, S and/or G2, is discussed.     

Different sensitivity of DNA in situ in interphase and metaphase chromatin to heat denaturation

Heat denaturation of DNA in situ, in unbroken cells, was studied in relation to the cell cycle. DNA in metaphase cells denatured at lower temperatures (8 degrees-10 degrees C lower) than DNA in interphase cells. Among interphase cells, small differences between G1, S, and G2 cells were observed at temperatures above 90 degrees C. The difference between metaphase and interphase cells increased after short pretreatment with formaldehyde, decreased when cells were heated in the presence of 1 mM MgCl2, and was abolished by cell pretreatment with 0.5 N HCl. The results suggest that acid-soluble constituents of chromatin confer local stability to DNA and that the degree of stabilization is lower in metaphase chromosomes than in interphase nuclei. These in situ results remain in contrast to the published data showing no difference in DNA denaturation in chromatin isolated from interphase and metaphase cells. It is likely that factors exist which influence the stability of DNA in situ are associated with the super-structural organization of chromatin in intact nuclei and which are lost during chromatin isolation and solubilization. Since DNA denaturation is assayed after cell cooling, there is also a possibility that the extent of denatured DNA may be influenced by some factors that control strand separation and DNA reassociation. The different stainability of interphase vs. metaphase cells, based on the difference in stability of DNA, offers a method for determining mitotic indices by flow cytofluorometry, and a possible new parameter for sorting cells in metaphase.     

Recognition of cells in mitosis by flow cytofluormetry.

Cells in mitosis may be distinguished from interphase cells based on difference in chromatin structure as revealed by two different methods of staining with acridine orange. In the first method, cells are heated and then stained at neutral pH; the difference in stainability between mitotic and interphase cells reflects the difference in the extent of deoxyribonucleic acid denatured by heat in these cells. At a given temperature the deoxyribonucleic acid of the mitotic cell appears to be more extensively denatured than that of the interphase cell. In the second method, cells are treated with buffer at pH 1.5 (1.3 to 1.9) and then stained at pH 2.6 (2.3 to 2.9). The mechanisms involved in the differential stainability of interphase versus mitotic cells at that low pH are currently under investigation. In both methods, in addition to enumerating cells in mitosis, it is possible to quantitate cells in G1, S and G2 phases of the cell cycle.     

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

The kinetics of isthmal cells in mouse antrum were examined in three ways: the duration of cell cycle and DNA-synthesizing (S) stage was measured by the 'fraction of labelled mitoses' method; the duration of interphase and mitotic phases was determined from how frequently they occurred; and 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 occurrence, 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.     

Threonine phosphorylation is associated with mitosis in HeLa cells

Phosphorylation and dephosphorylation of proteins play an important role in the regulation of mitosis and meiosis. In our previous studies we have described mitosis-specific monoclonal antibody MPM-2 that recognizes a family of phosphopeptides in mitotic cells but not in interphase cells. These peptides are synthesized in S phase but modified by phosphorylation during G2/mitosis transition. The epitope for the MPM-2 is a phosphorylated site. In this study, we attempted to determine which amino acids are phosphorylated during the G2-mitosis (M) transition. We raised a polyclonal antibody against one of the antigens recognized by MPM-2, i.e. a protein of 55 kDa, that is present in interphase cells but modified by phosphorylation during mitosis. This antibody recognizes the p55 protein in both interphase and mitosis while it is recognized by the monoclonal antibody MPM-2 only in mitotic cells. Phosphoamino acid analysis of protein p55 from 32P-labeled S-phase and M-phase HeLa cell extracts after immunoprecipitation with anti-p55 antibodies revealed that threonine was extensively phosphorylated in p55 during G2-M but not in S phase, whereas serine was phosphorylated during both S and M phases. Tyrosine was not phosphorylated. Identical results were obtained when antigens recognized by MPM-2 were subjected to similar analysis. As cells completed mitosis and entered G1 phase phosphothreonine was completely dephosphorylated whereas phosphoserine was not. These results suggest that phosphorylation of threonine might be specific to some of the mitosis-related events.     

S phase, an evolutionary chromatin condensation state from G1 to G2, in a breast epithelial cell line.

In order to better understand the changes in DNA organization during the cell cycle, we quantified the chromatin texture of breast epithelial cells and followed its evolution through a cell cycle. The diversity of quiescent cell states led us to limit this study to proliferating cell phases, and to choose a cell line with no G0 cells, the MDA AG cell line. We recently developed a methodology for characterizing in situ the cell cycle of breast epithelial cell lines using a cell image processor. This method is based on 15 densitometric and texture parameters computed on individual Feulgen-stained nuclei and on multiparametric analysis of the resulting data. Chromatin pattern assessment is based on nine texture parameters measured from grey-level co-occurrence and run-length section matrices. In the present study, texture parameter computation showed gradual and progressive modifications of nuclear texture. While discrimination of G1, G2 and M phases was possible, we could not discriminate G1 from S and S from G2. The chromatin pattern (defined by these nine parameters) in the G1 and early S phases, on the one hand, and in the late S and G2 phases, on the other hand, were similar. The parameter values of cells in the S phase progressively increased from G1 to G2. Two interphase chromatin condensation states were distinguished in these breast cells: a base state characteristic of a prereplicative stage and a very granular state characteristic of a postreplicative stage. We hypothesized that S cells are a blend of these two states, the evolution of a non-duplicated state toward a duplicated one.     

Demonstration of a reaggregation inhibitor in sea urchin embryos

We used a new method based on the study of nuclear areas above certain density thresholds to estimate changes in the condensation of chromatin of a cell. Allium cepa L. root meristematic cells were “labelled” as binucleate by a 1 h treatment with 0.1 % caffeine and were fixed at the middle of each interphase period. The distribution of chromatin densities of Feulgen-stained cells in G1, S and G2 phases was so different that by simply estimating chromatin patterns it would be possible to identify which period of the interphase any cell has reached. G2 nuclei have an increased number of chromatin-dense areas compared with G1 or S nuclei. We postulate that the estimation of chromatin condensation may be useful for the evaluation of intranuclear differentiation at the level of the intact cell.     

Characterizing the three-dimensional organization of telomeres.

BACKGROUND: Quantitative analysis can be used in combination with fluorescence microscopy. Although the human eye is able to obtain good qualitative results, when analyzing the spatial organization of telomeres in interphase nuclei, there is a need for quantitative results based on image analysis. METHODS: We developed a tool for analyzing three-dimensional images of telomeres stained by fluorescence in situ hybridization in interphase nuclei with DNA counterstained with 4',6-diamidino-2-phenylindole. After deconvolution of the image, we segmented individual telomeres. From the location of the telomeres we derived a distribution parameter rhoT, which indicated whether the telomeres were in a disk (rhoT > 1) or not (rhoT approximately 1). We sorted mouse lymphocyte nuclei and measured rhoT. We also performed a bromodeoxyuridine synchronous cell sorting experiment on live cells and measured rhoT at several instances. RESULTS: Measuring rhoT for nuclei in G0/G1, S, and G2 produced 1.4 +/- 0.1, 1.5 +/- 0.2, and 14 +/- 2, respectively, showing a significant difference between G2 and G0/G1 or S. For the bromodeoxyuridine synchronous cell sorting experiment, we found a cell cycle dependency of rhoT and a correlation between rhoT and an observer. CONCLUSIONS: In this study we present a quantitative method to characterize the organization of telomeres using three-dimensional imaging, image processing, and image analysis.     

Alteration of cell cycle progression in human leukemia cell line (KOPM-28) induced by 12-o-tetradecanoylphorbol-13-acetate

Terminal cell differentiation usually results in an irreversible arrest in the G1 phase of the cell cycle and loss of cell renewal ability. Human promyelocytic leukemia HL-60 cells induced with 12-o-tetradecanoylphorbol-13-acetate (TPA) differentiate into monocytes/macrophages and accumulate in G1. We determined the effect of TPA on the growth kinetics of a human leukemia cell line (KOPM-28), which developed several of the characteristics of megakaryocytes in response to TPA, such as the surface antigen complex IIb/IIIa, platelet peroxidase and polyploidy. Cell growth was immediately and completely inhibited by TPA. Flow cytometric analysis of cellular DNA content revealed a gradual decrease in cells in G1 and an accumulation of cells in G2. These data suggest that TPA prolonged G1 and rapidly arrested the cells in G2. Synchronized cells were utilized to further analyze the rapid G2 arrest. Cells arrested with aphidicolin at the G1/S interphase were released, and the effects of TPA (added at different intervals) on cell cycle progression were examined 14 h after release. The results showed that TPA added at the end of the S phase, as well as at the G1/S interphase incompletely but distinctly arrested cells in G2. Moreover, G2 arrest was observed when TPA was added to cells released from a colcemid-induced G2/M block, suggesting that cells already in G2 were inhibited by TPA from moving through M to G1. Since some cells became multi-nucleated in the course of incubation with TPA, this G2 accumulation may have resulted at least in part from a prolongation of the phase or a transient G2 block. These changes in cell cycle progression induced by TPA may be characteristic of and/or related to megakaryocytic differentiation of hemopoietic precursor cells.     

Effect of 2-mercaptoethanol on the individual periods of the mitotic cycle

Dynamics of the mitotic cycle of the KEPV cells being on different interphase stages at the start of a 20 hour 2-mercaptoethanol (0.001 M) treatment has been studied during the treatment and for 11 hours after washing out the agent. The KEPV cells affected by mercaptoethanol during the interphase (G1, S, G2) were shown to continue their passage through the cycle to enter mitosis, but part of the cells of the S period and of the first half of the G2 period were arrested in the interphase. In the presence of mercaptoethanol, mitotic cells reach the metaphase stage, and their further behaviour depends on the duration of the treatment. For the first 8 hours of treatment, a phase of "unstable block" exists for cells that were in S and G2 periods at the beginning of treatment, while other cells are transformed into K-metaphases. 8 hours later a phase of "stable block" occurs and all the normal metaphases are transformed into K-metaphases. After washing out the culture from mercaptoethanol the cells are ejected from the block in K-metaphase. The transformation from K-metaphase into the normal metaphase is realised in the course of this process. The cells which were in S and G2 periods at the beginning of the treatment are ejected from the block simultaneously after washing, while the cells of the G1 period--with a small delay. After washing out mercaptoethanol the cells that were in the interphase (G1, S, G2) at the beginning of the treatment are capable of producing both multipolar mitoses and mitoses without cytotomy.     

BM28, a human member of the MCM2-3-5 family, is displaced from chromatin during DNA replication

We have recently cloned and characterized a human member (BM28) of the MCM2-3-5 family of putative relication factors (Todorov, I.T., R. Pepperkok, R.N. Philipova, S. Kearsey, W. Ansorge, and D. Werner. 1994. J. Cell Sci. 107:253-265). While this protein is located in the nucleus throughout interphase, we report here a dramatic alteration in its nuclear binding during the cell cycle. BM28 is retained in the nucleus after Triton X-100 extraction in G1 and early S phase cells, but is progressively lost as S phase proceeds, and little BM28 is retained in detergent-extracted G2 nuclei. BM28 that is resistant to extraction in G1 nuclei is removed by DNase I digestion, suggesting that the protein is chromatin associated. In addition, we present evidence for variations in the electrophoretic mobility of BM28 that may reflect posttranslational modifications of BM28 during the cell cycle. During mitosis, BM28 is present as a fast-migrating form, but on entry into G1, the protein is converted into a slow-migrating form. With the onset of S phase, the slow-migrating form is progressively converted into the fast form. BM28 is phosphorylated at all stages of the cell cycle, but during interphase the fast form is hyperphosphorylated compared with the slow form. These apparent changes in modification may reflect or effect changes in the nuclear binding of BM28. The behavior of BM28 is not dissimilar to related proteins in Saccharomyces cerevisiae, such as Mcm2p, which are excluded from the nucleus after DNA replication. We speculate that BM28 may be involved in the control that limits eukaryotic DNA replication to one round per cell cycle.     

Haspin inhibition delays cell cycle progression through interphase in cancer cells

Haspin (Haploid Germ Cell-Specific Nuclear Protein Kinase) is a serine/threonine kinase pertinent to normal mitosis progression and mitotic phosphorylation of histone H3 at threonine 3 in mammalian cells. Different classes of small molecule inhibitors of haspin have been developed and utilized to investigate its mitotic functions. We report herein that applying haspin inhibitor CHR-6494 or 5-ITu at the G1/S boundary could delay mitotic entry in synchronized HeLa and U2OS cells, respectively, following an extended G2 or the S phase. Moreover, late application of haspin inhibitors at S/G2 boundary is sufficient to delay mitotic onset in both cell lines, thereby, indicating a direct effect of haspin on G2/M transition. A prolonged interphase duration is also observed with knockdown of haspin expression in synchronized and asynchronous cells. These results suggest that haspin can regulate cell cycle progression at multiple stages at both interphase and mitosis.     

The centriolar cycle in polykaryons containing prematurely condensed chromosomes or telophase-like nuclei]

In fused interphase-mitotic cells, either interphase nuclei are induced to premature chromosome condensation (PCC) or mitotic chromosomes are induced to telophase-like nuclei (TLN) formation. This study concerns structural and functional changes in centrioles of fused cells in which PCC or TLN are induced. Embryonic pig kidney cells were fused using a modified PEG-DMSO-serum method. Cell cycle period of the nuclei was determined before cell fusion using double-labeling autoradiography. Polykaryons containing desirable type of PCC or interphase nuclear combination in TLN were selected on the basis of isotope labeling after being embedded in epon. Selected cells were cut into serial sections and studied under electron microscope. The data obtained showed that centrioles at every interphase period undergo mitotic activation when their nuclei are induced to PCC. They acquire fibrillar halo and form half-spindles. Daughter centrioles at G1, S and G2 periods are also capable of mitotic activation when separated from their mother centriole. Inert centrioles were found in some cells with G1-PCC. When mitotic nuclei are induced to TLN formation, their centrioles also become inactivated. They lose fibrillar halo and mitotic spindles break down. Some mitotic centrioles develop features characteristic of interphase period such as satellites and vacuoles. Induced nuclear and centriolar changes are simultaneous and may be controlled by the same factor. Mitotic factor of mitotic cell partner which induces PCC may also induce interphase centrioles to mitotic activation. Degradation of the mitotic factor leading to TLN formation may also cause the loss of the mitotic activity of centrioles and disorganization of mitotic spindles.     

微核形成与细胞周期关系的初步研究——Ⅳ.化学诱变剂诱发人淋巴细胞间期各阶段的微核形成

本实验应用具有诱变作用的抗癌药:噻地哌、长春新碱,乙双吗啉等,体内或体外处理诱发人体外周血淋巴细胞微核,通过控制细胞培养时间,放射性自显影及中期细胞阻滞等方法,定量地分析了细胞间期各阶段的微核率(MNF)。本组实验结果表明,间期各阶段均可有不同程度的微核形成,其中最多的是G_1期,其次是G_2期和G_0期。S期细胞的MNF较G_1期有极显著的下降,这提示大部分G_1期的微核细胞不能进入S期,使细胞增殖中止,这可能是抗癌药物杀伤肿瘤细胞的机制之一。     

Cell cycle variations in chromatin structure detected by DNase I

We have recently developed a reproducible method for the use of DNase I as a sensitive probe of chromatin structure (Prentice, D A & Gurley, L R, Biochim biophys acta 740 (1983) 134) [12] and have used this probe to investigate chromatin structure during the interphase of the cell cycle. Chinese hamster cells (line CHO) were synchronized by: (1) mitotic detachment, to obtain M-phase cells; (2) isoleucine deprivation, to obtain G1-phase cells; and (3) sequential use of isoleucine deprivation followed by release into the presence of hydroxyurea, to obtain cells blocked at the start of S phase. The cells were released from the various blocking schemes and nuclei were isolated and digested with DNase I at various times. The digestion kinetics were monitored to detect possible changes in chromatin condensation through the cell cycle. The chromatin was much more accessible to DNase I in G1 phase than in S or G2 phase, with only small variations in structure detected in late G1 and very early S phase. From early S phase up to mitosis, the chromatin became increasingly condensed and inaccessible to DNase I action. These results support the concept of a chromatin condensation cycle during interphase as well as during mitosis.     

Synchronisation of cell division in the shoot apex of Silene in relation to flower initiation

In plants of Silene coeli-rosa, induced to flower by 7 LD, synchronisation of cell division in 20 per cent or more of the cells in the shoot apical dome was found on the 8th and 9th days after the beginning of induction, during the plastochron before sepal initiation. Synchronisation was inferred from the changes in the proportions of cells with the 2C and 4C amounts of DNA, and changes in mitotic index and labelling index. From the peaks of mitotic index a cell cycle of 10 h was measured for the synchronised cells, half that of cells in the apices of uninduced plants in short days. The faster cell cycle and synchronisation in the induced plants was associated with a shortening, of both G1 and G2, suggesting two control points, while S and M remained unchanged. These results are compared with those from other plants in which synchronisation occurs at the beginning rather than the end of evocation.Abbreviations LD long day(s) - SD short day(s) - S DNA synthesis phase of cell cycle - G1 pre-S interphase - G2 post-S interphase - M mitosis     

MPF Induction of Microtubule Assembly at Interphase Kinetochore of CHO Cells

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The durations and compositions of cell cycles in embryos of the leech, Helobdella triserialis

When tritiated thymidine triphosphate ([(3)H]TTP) or its immunohistochemically detectable analogue, bromodeoxyuridine triphosphate (BrdUTP), is injected into blastomeres of leech embryos it passes throughout the entire embryo and is rapidly incorporated (within 2 min after injection) into nuclei of cells synthesizing DNA (S phase). In the same embryos a DNA-specific stain can be used to identify cells in mitosis (M phase) or nonreplicative interphase (G(1) or G(2) phase) on the basis of nuclear or chromosomal morphology. Using this procedure, we have determined the lengths and compositions of the mitotic cell cycles of identifiable cells in early embryos of the leech, Helobdella triserialis, and have analysed how the cell cycles change during the first seven stages of development. The relatively short cell cycles of the early blastomeres comprise not only phases of M and S, but also postreplicative gap (G(2)) phases. The lengthening of the cell cycles that occurs as development progresses is primarily accomplished by an increase in the length of G(2) and secondarily by an increase in the length of S and,in some instances, the addition of a prereplicative gap(G(1)) phase; M phase remains relatively constant. These data suggest that the durations of the cell cycles of embryonic cells are regulated by a variety of mechanisms.