Application of pyronin Y(G) in cytochemistry of nucleic acids

Chinese hamster ovary (CHO) cells or isolated nuclei were stained with pyronin Y(PY) and analyzed by absorption or fluorescence microscopy, as well as by flow cytometry. Specificity of the staining reaction was assayed by testing sensitivity of the stainable material to RNase or DNase. The colored complexes detected by light absorption in fixed cells stained with PY are nonfluorescent and are most likely the products of condensation of single-stranded (ss) RNA by PY; the poly(rA) and poly(rA,rG) are the most sensitive to condensation. The products of PY interaction with double-stranded (ds) nucleic acids are fluorescent and can be detected in cells by cytofluorometry. PY used alone stains both DNA and RNA, and the staining capabilities of these nucleic acids vary depending upon the PY concentration at equilibrium; at a concentration above 330 microM, the RNA stainability decreases, perhaps due to its denaturation and condensation caused by the dye. In the presence of Hoechst 33342, PY can specifically stain RNA in fixed cells or isolated cell nuclei. Because only complexes of PY with ds RNA are fluorescent, this dye can be used as a probe of RNA conformation, e.g., to monitor denaturation of RNA in situ. The RNA stainability of mitotic cells is about 25% lower than that of cells in G2 phase, which indicates that during mitosis proportionately less cellular RNA is in the ds conformation. The advantages and limitations of the two cytochemical methods for DNA/RNA detection, one based on the use of Hoechst 33342 and PY, and another employing the metachromatic properties of acridine orange, are compared.     

Caffeine increases sensitivity of DNA to denaturation in chromatin of L1210 cells.

Exposure of exponentially growing L1210 cells to 5 mM and higher concentrations of caffeine perturbs their progression through the cell cycle and results in increased sensitivity of DNA in situ to denaturation. The latter is detected by the increased metachromatic stainability of DNA with acridine orange (AO) and sensitivity to S1 nuclease, measured by flow cytometry. Decreased DNA stability is generally characteristic of chromatin condensation and in untreated cells is observed in mitosis or quiescence (G0). The caffeine-induced decrease in DNA stability affects the interphase cells regardless of their position in the cycle and the changes are stochastic, concentration- and time-dependent. Populations of cells responding to caffeine are very heterogenous with respect to the degree of destabilization of DNA; sensitivity of DNA to denaturation of the maximally affected cells is similar to that of untreated cells in mitosis. The present method allows one to quantitatively express effects of caffeine on nuclear chromatin in individual cells of large cell populations and may be employed in studies correlating chromatin changes induced by this agent with its effects in modulation of cell sensitivity to radiation or antitumour drugs.     

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.     

Caffeine increases sensitivity of DNA to denaturation in chromatin of L1210 cells

Abstract.
Exposure of exponentially growing L1210 cells to 5 mM and higher concentrations of caffeine perturbs their progression through the cell cycle and results in increased sensitivity of DNA in situ to denaturation. The latter is detected by the increased metachromatic stainability of DNA with acridine orange (AO) and sensitivity to S₁ nuclease, measured by flow cytometry. Decreased DNA stability is generally characteristic of chromatin condensation and in untreated cells is observed in mitosis or quiescence (G₀). The caffeine-induced decrease in DNA stability affects the interphase cells regardless of their position in the cycle and the changes are stochastic, concentration- and time-dependent. Populations of cells responding to caffeine are very heterogenous with respect to the degree of destabilization of DNA; sensitivity of DNA to denaturation of the maximally affected cells is similar to that of untreated cells in mitosis. The present method allows one to quantitatively express effects of caffeine on nuclear chromatin in individual cells of large cell populations and may be employed in studies correlating chromatin changes induced by this agent with its effects in modulation of cell sensitivity to radiation or antitumour drugs.     

Condensed chromatin and cell inactivation by single-hit kinetics

Mammalian cells are extremely sensitive to gamma rays at mitosis, the time at which their chromatin is maximally condensed. The radiation-induced killing of mitotic cells is well described by single-hit inactivation kinetics. To investigate if radiation hypersensitivity by single-hit inactivation correlated with chromatin condensation, Chinese hamster ovary (CHO) K1 (wild-type) and xrs-5 (radiosensitive mutant) cells were synchronized by mitotic shake-off procedures and the densities of their chromatin cross sections and their radiosensitivities were measured immediately and 2 h into G1 phase. The chromatin of G1-phase CHO K1 cells was dispersed uniformly throughout their nuclei, and its average density was at least three times less than in the chromosomes of mitotic CHO K1 cells. The alpha-inactivation co-efficient of mitotic CHO K1 cells was approximately 2.0 Gy(-1) and decreased approximately 10-fold when cells entered G1 phase. The density of chromatin in CHO xrs-5 cell chromosomes at mitosis was greater than in CHO K1 cell chromosomes, and the radiosensitivity of mitotic CHO xrs-5 cells was the greatest with alpha = 5.1 Gy(-1). In G1 phase, CHO xrs-5 cells were slightly more resistant to radiation than when in mitosis, but a significant proportion of their chromatin was found to remain in condensed form adjacent to the nuclear membrane. These studies indicate that in addition to their known defects in DNA repair and V(D)J recombination, CHO xrs-5 cells may also be defective in some process associated with the condensation and/or dispersion of chromatin at mitosis. Their radiation hypersensitivity could result, in part, from their DNA remaining in compacted form during interphase. The condensation status of DNA in other mammalian cells could define their intrinsic radiosensitivity by single-hit inactivation, the mechanism of cell killing which dominates at the dose fraction size (1.8-2.0 Gy) most commonly used in radiotherapy.     

RIBONUCLEIC ACID AND PROTEIN SYNTHESIS IN MITOTIC HELA CELLS

HeLa cells arrested in mitosis were obtained in large numbers, with only very slight interphase cell contamination, by employing the agitation method of Terasima and Tolmach, and Robbins and Marcus. Protein synthesis and RNA synthesis were almost completely suppressed in mitotic cells. Active polyribosomes were nearly absent in mitotic cells as compared with interphase cells treated in the same way. Cell-free protein synthesis and RNA polymerase activity were also greatly depressed in extracts of metaphase cells. The deoxyribonucleoprotein (DNP) of condensed chromosomes from mitotic cells was less efficient as a template for Escherichia coli RNA polymerase than was DNP from interphase cells, although isolated DNA from both sources was equally active as a primer. Despite very poor endogenous amino acid incorporation by extracts of metaphase cells, polyuridylate stimulated phenylalanine incorporation by a larger factor in mitotic cell extracts than it did in interphase cell extracts. These results suggest that RNA synthesis is suppressed in mitotic cells because the condensed chromosomes cannot act as a template, and that protein synthesis is depressed at least in part because messenger RNA becomes unavailable to ribosomes. This conclusion was supported by the demonstration that cells arrested in metaphase supported multiplication of normal yields of poliovirus, thereby showing that the mitotic cell is capable of considerable synthesis of RNA and protein.     

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.     

The induction of chromosomal aberrations and SCEs by visible light in combination with dyes. II. Cell cycle dependence, and the effect of hydroxyl radical scavengers during light exposure in cultures of Chinese hamster ovary cells sensitized with acridine orange

Chinese hamster ovary (CHO) cells were synchronized by mitotic shake-off, treated with the fluorochrome acridine orange (AO; 0.5 micrograms/ml), washed free of excess dye and subsequently exposed to visible light (2 X 40 W/8 Wm-2). The light exposure was performed on cells in the G1, G1/S, S or G2 phase of the cell cycle. AO + light induced high frequencies of aberration in the S phase and even higher in the G1 phase. The aberrations observed were all of the chromatid type. The chromosome-type aberrations (dicentrics, rings) obtained when cells in the G1 phase were exposed to X-rays were not found after corresponding treatments with AO + light. With the exception of an increased frequency of gaps, no chromosomal aberrations were induced in G2-phase cells. Sister-chromatid exchanges were efficiently produced by the photodynamic system in the G1, G1/S and S phase of the cell cycle. In other experiments, AO-treated unsynchronized CHO cells were exposed to light in the presence of the hydroxyl radical scavengers mannitol (100 mM) and 5-dimethyl thiourea (100 mM). In parallel experiments these scavengers were found to reduce markedly the chromosome breaking effects by X-rays but had no influence on the photodynamic induction of chromosomal alterations. The results presented show that the visible light-induced chromosomal alterations in CHO cells sensitized with the fluorochrome AO are obtained by an S-dependent mechanism. Furthermore, the results indicate that the hydroxyl free radical does not play a major role in the production of chromosomal alterations by AO + light.     

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.     

Chromosome architecture can dictate site-specific initiation of DNA replication in Xenopus egg extracts

Xenopus egg extracts initiate DNA replication specifically at the dihydrofolate reductase (DHFR) origin locus with intact nuclei from late G1-phase CHO cells as a substrate, but at nonspecific sites when purified DNA is assembled by the extract into an embryonic nuclear structure. Here we show that late G1-phase CHO nuclei can be cycled through an in vitro Xenopus egg mitosis, resulting in the assembly of an embryonic nuclear envelope around G1-phase chromatin. Surprisingly, replication within these chimeric nuclei initiated at a novel specific site in the 5' region of the DHFR structural gene that does not function as an origin in cultured CHO cells. Preferential initiation at this unusual site required topoisomerase II-mediated chromosome condensation during mitosis. Nuclear envelope breakdown and reassembly in the absence of chromosome condensation resulted in nonspecific initiation. Introduction of condensed chromosomes from metaphase- arrested CHO cells directly into Xenopus egg extracts was sufficient to elicit assembly of chimeric nuclei and preferential initiation at this same site. These results demonstrate clearly that chromosome architecture can determine the sites of initiation of replication in Xenopus egg extracts, supporting the hypothesis that patterns of initiation in vertebrate cells are established by higher order features of chromosome structure.     

Changes in nuclear chromatin related to apoptosis or necrosis induced by the DNA topoisomerase II inhibitor fostriecin in MOLT-4 and HL-60 cells are revealed by altered DNA sensitivity to denaturation.

The antitumor drug fostriecin (phosphotrienin, FST) has been reported to exert its cytostatic and cytotoxic effects via inhibition of DNA topoisomerase II. The sensitivity of human lymphocytic leukemic MOLT-4 and promyelocytic HL-60 leukemic cells to a wide range of FST concentrations was studied by analyzing the cell cycle-specific effects and changes in nuclear chromatin induced by this inhibitor. The latter was evaluated by assaying the sensitivity of DNA in situ to acid-induced denaturation cytofluorimetrically, with the use of the metachromatic fluorochrome acridine orange (AO), which differentially stains double-stranded and denatured DNA. The cytostatic effects were observed soon after addition of FST (at concentrations of 1-30 microM for MOLT-4 cultures and 1-5 microM for HL-60 cultures) as a perturbation of cell progression through S and G2 phases of the cell cycle. Cell progression through the cycle was halted at greater than 30 microM FST in MOLT-4 cultures and at greater than 5 microM in HL-60 cultures; the effect was instantaneous and affected all phases of the cycle, so that no changes in the cell cycle distribution were apparent with increasing length of exposure to the drug. Instead, at these high FST concentrations, immediate cytotoxic effects became evident, manifesting either as cell apoptosis or necrosis. Apoptosis was observed only in the case of HL-60 cells, at FST concentrations of 5-100 microM, and was characterized by markedly increased sensitivity of DNA to denaturation combined with a decrease in overall DNA stainability, either with the DNA-specific dye DAPI or with AO, indicative of the activation of endogenous nucleases. Necrotic cell death was observed at FST concentrations of 1 mM and at greater than 30 microM for HL-60 and MOLT-4 cells, respectively: in both cases the overall DNA stainability, with either DAPI or AO, was unchanged compared to the control, but their DNA was very sensitive to denaturation. Interestingly, DNA in G2 and late S phase MOLT-4 cells, which were undergoing necrotic death, was much more sensitive to denaturation than was DNA in G1 cells of this lineage. The data indicate that chromatin changes induced by DNA topoisomerase II inhibitors in cells that undergo apoptotic or necrotic death can be conveniently monitored by the assay of DNA in situ sensitivity to denaturation.     

Flow cytometric detection of mitotic cells using the bromodeoxyuridine/DNA technique in combination with 90 degrees and forward scatter measurements

Mitotic cells could be well discriminated from the cells in the G1-, S- and G2-phases of the cell cycle using pulse labeling of S-phase cells with bromodeoxy-uridine (BrdUrd) and staining of the cells for incorporated BrdUrd and total DNA content. Unlabeled G2- and M-phase cells could be measured as two separate peaks according to propidium iodide fluorescence. M-phase cells showed lower propidium iodide fluorescence emission compared to G2-phase cells. The fluorescence difference of M- and G2-phase cells was caused by the different thermal denaturation of their DNA. Best separation of M- and G2-phase cells was obtained after 30-50 min heat treatment at 95 degrees C. Mitotic index could be measured if no unlabeled S-phase cells were present in the cell culture. With additional measurements of 90 degree scatter and/or forward scatter signals, mitotic cells could be clearly discriminated from both unlabeled G2- and S-phase cells. The correct discrimination (about 99%) of mitotic cells from interphase cells was verified by visual analysis of the nuclear morphology after selective sorting. Unlabeled and labeled mitotic cells could be observed as pulse-labeled cells progressed through the cell cycle. We conclude that this modified BrdUrd/DNA technique using prolonged thermal denaturation and the simultaneous measurement of scatter signals may offer additional information especially in the presence of BrdUrd-unlabeled S-phase cells.     

Correlation between cell cycle duration and RNA content.

The metachromatic fluorochrome acridine orange was used to differentially stain DNA and RNA in Chinese hamster ovary (CHO) cells and in mitogen-stimulated human lymphocytes during their progression through the cell cycle. Green and red fluorescence of individual cells, representing cellular DNA and RNA, respectively, was measured by flow cytometry. CHO cells were synchronized by selective detachment at mitosis. Their rate of progression through G1 and subsequently through S phase correlated with the content of stainable RNA. The mean duration of the G1 phase was 5.2 hours for cells with high RNA content (highest 25 percentile population) and 8.1 hours for cells with low RNA (lowest 25 percentile). The duration of S phase was 5.9 and 7.5 hours for high- and low-RNA, 25 percentile subpopulations, respectively. Lymphocytes synchronized at the G1/S boundary by hydroxyurea or 5-fluorodeoxyuridine showed extremely high intercellular variation with respect to content of stainable RNA. After release from the block they traversed S phase at rates linearly proportional to the content of stainable RNA. The duration of S phase was five hours for cells with high RNA-, six to nine hours for cells with moderate RNA- and up to 27 hours for cells with minimal RNA-content. The data suggest that the rate of progression through the cell cycle of individual cells within a population may be correlated with the number of ribosomes per cell.     

Characterization of subcellular components in synchronized hepatoma cells as a function of the cell cycle

The specific activity and subcellular distribution of marker enzymes for the main subcellular components were analysed in homogenates of synchronized hepatoma cells (Morris 7288c), obtained by selective detachment at mitosis combined with a metaphase block with Colcemid. Markers for lysosomes, mitochondrial outer membrane, plasma membrane and cytosol are synthesized throughout the cycle at the same rate as the bulk of cellular protein. Larger variations are observed for a Golgi marker; after a decrease around mitosis, the specific activity of galactosyltransferase increases steadily from middle G(1)-phase on, and at the end of G(2)-phase it is nearly twice that observed at the beginning of G(1)-phase. Our results show that synthesis of cytochrome oxidase may occur preferentially in G(2)-phase. Large modifications of the density distribution of lysosomes are observed during the cell cycle; the median equilibrium density of lysosomal markers decreases in G(1)-phase, and some increase in soluble activity occurs at the same time. Reverse changes occur progressively during S- and G(2)-phases. At mitosis, Golgi galactosyltransferase shows a more dispersed distribution, and modifications in the density distribution of endoplasmic-reticulum NADPH-cytochrome c reductase are observed. The latter can be most easily explained by a detachment of ribosomes from endoplasmic-reticulum membranes. No significant modifications occur in mitochondrial and plasma-membrane markers.     

Ultrastructural labeling of cell surface lectin receptors during the cell cycle.

The distribution of specific surface receptors in the course of the cell cycle has been studied on two transformed cell lines by means of ultrastructural labelling techniques employing Concanavalin A (ConA) and wheat germ agglutinin (WGA). Synchronized cultures of Cl₂TSV₅, an SV40-transformed hamster cell line and of CHO cells were labelled as monolayers or in suspension in the different phases of the cell cycle. In cells labelled in monolayers, a moderately discontinuous pattern of surface labelling was present during G 1, S, and G 2. On cells in mitosis, however, this pattern changes strikingly and becomes very discontinuous. These results indicate that the degree of receptor clustering is greater in mitosis than in interphase. In cells labelled in suspension, the differences in pattern between mitosis and interphase were absent. Colcemid treatment did not modify the distribution of the label, either in interphase or in mitosis. Moreover, cells in mitosis collected by Colcemid treatment and labelled at a moment in which parallel unblocked cultures had completed mitosis and passed into G 1 showed an interphase-type labelling pattern; this indicates that a certain dissociation exists between surface events and nuclear events during mitosis. These results are discussed in terms of several factors that may contribute to the production of receptor clustering, namely, direct lectin action, surface movement and membrane flow, participation of cytoplasmic structures and, finally, attachment of cells to a substratum.     

Hydroxyurea retards the progression of G2 cells

Continuous treatment of monolayer cultures of Chinese hamster ovary (CHO) cells with 1.0 mM HU reduced the rate at which G2 cells entered mitosis. Although all cells in G2+M at the start of treatment eventually entered mitosis, early G2 cells, which were exposed to HU the longest, entered mitosis 45 min later than untreated G2 cells. This perturbation of G2 progression should be considered when employing HU as a synchronizing agent in mammalian cell populations.     

Differences in sequence content of nuclear and cytoplasmic polyribosomal RNA from adenovirus-infected cells.

RNA molecules from nuclear and cytoplasmic polyribosomes of adenovirus-infected HeLa cells were compared by hybridization to analyse the sequence content. Nuclear polyribosomes were released by exposure of intact detergent-washed nuclei to poly(U) and purified. Cytoplasmic polyribosomes were also purified from the same cells. To show that nuclear polyribosomes contain ribosomes linked by mRNA, polyribosomes were labelled with methionine and uridine in the presence of actinomycin D in adenovirus-infected cells. Purified nuclear polyribosomes were treated with EDTA under conditions which dissociate polyribosomes into ribosomes and subunits with a simultaneous release of mRNA, and sedimented. The treatment dissociated these polyribosomes, releasing the mRNA from them. Radiolabelled total RNA from each polyribosome population was fractionated in sucrose gradients into several pools or hybridized to intact adenovirus DNA to select virus-specific RNA. Sucrose-gradient-fractionated pool-3 RNA (about 28S) and virus-specific RNA were then hybridized to fragments of adenovirus DNA cleaved by restriction endonucleases SmaI, HindIII and EcoRI by the Southern-blot technique and by filter hybridization. The results showed that nuclear RNA contained sequences, from about 0 to 18 map units, which were essentially absent from cytoplasmic RNA. Furthermore, the amount of virus-specific RNA for a particular sequence was also different in the two populations.     

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.     

Inactivation of Cdk1/Cyclin B in metaphase-arrested mouse FT210 cells induces exit from mitosis without chromosome segregation or cytokinesis and allows passage through another cell cycle

It is well known that inactivation of Cdk1/Cyclin B is required for cells to exit mitosis. The work reported here tests the hypothesis that Cdk1/Cyclin B inactivation is not only necessary but also sufficient to induce mitotic exit and reestablishment of the interphase state. This hypothesis predicts that inactivation of Cdk1 in metaphase-arrested cells will induce the M to G1-phase transition. It is shown that when mouse FT210 cells (in which Cdk1 is temperature-sensitive) are arrested in metaphase and then shifted to their non-permissive temperature, they rapidly exit mitosis as evidenced by reassembly of interphase nuclei, decondensation of chromosomes, and dephosphorylation of histones H1 and H3. The resulting interphase cells are functionally normal as judged by their ability to progress through another cell cycle. However, they have double the normal number of chromosomes because they previously bypassed anaphase, chromosome segregation, and cytokinesis. These results, taken together with other observations in the literature, strongly suggest that in mammalian cells, inactivation of Cdk1/cyclin B is the trigger for mitotic exit and reestablishment of the interphase state.     

In situ factors affecting stability of the DNA helix in interphase nuclei and metaphase chromosomes

The data from earlier cytochemical studies, in which the metachromatic fluorochrome acridine orange (AO) was used to differentially stain single vs double-stranded DNA, suggested that DNA in situ in intact metaphase chromosomes or in condensed chromatin of G0 cells is more sensitive to denaturation, induced by heat or acid, than DNA in decondensed chromatin of interphase nuclei. Present studies show that, indeed, DNA in permeabilized metaphase cells, in contrast to cells in interphase, when exposed to buffers of low pH (1.5-2.8) becomes digestible with the single-strand-specific S1 or mung bean nucleases. A variety of extraction procedures and enzymatic treatments provided evidence that the presence of histones, HMG proteins, and S-S bonds in chromatin, as well as phosphorylation or poly(ADP)ribosylation of chromatin proteins, can be excluded as a factor responsible for the differential sensitivity of metaphase vs interphase DNA to denaturation. Cell treatment with NaCl at a concentration of 1.2 N and above abolished the difference between interphase and mitotic cells, rendering DNA in mitotic cells less sensitive to denaturation; such treatment also resulted in decondensation of chromatin visible by microscopy. The present data indicate that structural proteins extractable with greater than or equal to 1.2 N NaCl may be involved in anchoring DNA to the nuclear matrix or chromosome scaffold and may be responsible for maintaining a high degree of chromatin compaction in situ, such as that observed in metaphase chromosomes or in G0 cells. Following dissociation of histones, the high spatial density of the charged DNA polymer may induce topological strain on the double helix, thus decreasing its local stability; this can be detected by metachromatic staining of DNA with AO or digestion with single-strand-specific nucleases.