Cell cycle-specific changes in histone phosphorylation associated with cell proliferation and chromosome condensation.

Preparative polyacrylamide gel electrophoresis was used to examine histone phosphorylation in synchronized Chinese hamster cells (line CHO). Results showed that histone f1 phosphorylation, absent in G(1)-arrested and early G(1)-traversing cells, commences 2 h before entry of traversing cells into the S phase. It is concluded that f1 phosphorylation is one of the earliest biochemical events associated with conversion of nonproliferating cells to proliferating cells occurring on old f1 before synthesis of new f1 during the S phase. Results also showed that f3 and a subfraction of f1 were rapidly phosphorylated only during the time when cells were crossing the G(2)/M boundary and traversing prophase. Since these phosphorylation events do not occur in G(1), S, or G(2) and are reduced greatly in metaphase, it is concluded that these two specific phosphorylation events are involved with condensation of interphase chromatin into mitotic chromosomes. This conclusion is supported by loss of prelabeled (32)PO(4) from those specific histone fractions during transition of metaphase cells into interphase G(1) cells. A model of the relationship of histone phosphorylation to the cell cycle is presented which suggests involvement of f1 phosphorylation in chromatin structural changes associated with a continuous interphase "chromosome cycle" which culminates at mitosis with an f3 and f1 phosphorylation-mediated chromosome condensation.

Nuclease digestibility of chromatin is affected by nuclei isolation procedures.

Experiments using nucleases as probes of chromatin structure take place in two stages: (1) nuclei isolation, and (2) nuclease digestion. The parameters of the nuclease digestion stage are usually strictly controlled because of nuclease sensitivity to them. However, there have been no reports on whether parameters in the nuclei isolation stage affect the subsequent nuclease digestions. We have evaluated a typical nuclei isolation technique with respect to how changes in the isolation parameters affect nuclease digestion kinetics. Our observations point out that various parameters encountered in the nuclei isolation stage have a significant effect on the subsequent nuclease digestion kinetics of DNAase I. These parameters include the concentration of cells, divalent cations and phosphatase inhibitors. The pH, concentration of NaCl and concentration of detergent had little effect. Micrococcal nuclease was relatively unaffected by changes in the nuclei isolation parameters. The importance of this report lies in the demonstration that lack of control of seemingly insignificant parameters, such as cell concentration during the nuclei isolation stage, leads to subsequent irreproducible results in the DNAase I digestion. These findings indicate that great care must be exercised in the nuclei isolation stage if reproducible work is to be performed with DNAase I.

Action of heparin on mammalian nuclei. II. Cell-cycle-specific changes in chromatin organization correlate temporally with histone H1 phosphorylation.

The interaction of the polyanion heparin with the inner histones of chromatin has been used to detect changes in chromatin organization associated with cell-cycle traverse. Synchronized populations of Chinese hamster cells were obtained either in early G1 or near the G1/S boundary. The rate of interaction of heparin with chromatin-associated inner histones was measured using nuclei isolated from synchronized cell populations in different phases of the cell cycle. A G1-specific decrease in rate of interaction of heparin with inner histones was observed and found to be independent of the presence of hydroxyurea during traverse of G1. A further decrease in heparin-inner histone interaction occurred in late S and G2. These changes correlate temporally with the interphase phosphorylation(s) of histone H1. This correlation is discussed within the framework of current models of higher order chromatin structure (i.e. organization above the nucleosome level). Analysis of the cooperativity of interaction of heparin with inner histones was performed using the kinetic analog of the Hill equation. This analysis suggests that the organization of inner histones on chromatin does not undergo large variations during the cell cycle.

Action of heparin on mammalian nuclei. I. Differential extraction of histone H1 and cooperative removal of histones from chromatin.

Heparin interacts strongly with the histone component of chromatin, forming heparin-histone complexes which resist dissociation by 0.2 M H2SO4. Heparin treatment of unfractionated histones isolated from nuclei of Chinese hamster cells indicates that the affinities of the histone classes for heparin appear in the order from greatest to least: (H3, H4) greater than (H2A, H2B) greater than H1. However, when isolated nuclei are treated with heparin, H1 is released from the chromatin more readily than the other four histone classes. The release of these four histones (H2A, H2B, H3, and H4) is coordinate and occurs in a highly cooperative manner, as indicated by (1) dependence of the initial kinetics of histone removal upon heparin concentration, (2) analysis of DNA and histones in the fractions obtained from differential sedimentation of heparin-treated nuclei, and (3) analysis of the products from heparin-treated nuclei by equilibrium centrifugation in metrizamide density gradients. The results suggest rapid procedures for using heparin as an agent for studying the accessibility of histones in chromatin of intact nuclei. The relationship of these results to current models of chromatin structure is discussed.

DNAase I and cellular factors that affect chromatin structure.

The use of DNAase I as a probe of chromatin structure is frequently fraught with problems of irreproducibility. We have recently evaluated this procedure, documented the sources of the problems, and standardized the method for reproducible results (Prentice and Gurley (1983) Biochim. Biophys. Acta 740, 134-144). We have now used this probe to detect differences in chromatin structure between cells blocked (1) in G1 phase by isoleucine deprivation, or (2) in early S phase by sequential use of isoleucine deprivation followed by release into the presence of hydroxyurea. The cells blocked in G1 phase have easily-digestible chromatin, while cells blocked in early S phase have chromatin which is much more resistant to DNAase I. These differences were found to be the result of diffusible factors found in the cytoplasm and nuclei of G1- and S-phase cells, respectively. The G1 cells contained a cytoplasmic factor which modulates the chromatin structure of S-phase nuclei to a more easily digestible state, while cells blocked in S phase contain a nuclear factor which modulates the chromatin structure of G1 nuclei to a state more resistant to digestion. DNAase I is much more sensitive to these cell cycle-specific chromatin changes than is micrococcal nuclease. The results indicate that, under controlled conditions, DNAase I should be a valuable probe for detecting chromatin structural changes associated with cell cycle traverse, differentiation, development, hormone action and chemical toxicity.

Composition and synthesis during G1 and S phase of a high mobility group-E/G component from Chinese hamster ovary cells.

A perchloric acid soluble protein from the sedimented chromatin of blended Chinese hamster ovary (line CHO) cells has been isolated by guanidine hydrochloride gradient chromatography on Bio . Rex-70 ion exchange resin. The amino acid composition of the protein (designated as CHO HMG-E/G) is similar to that of mouse HMG-E, but it differs from that of bovine HMG-14 and HMG-17 or any possible mixture of the two. CHO HMG-E/G incorporates [32P]phosphate like HMG-14 and HMG-17 class proteins from other species, but all resolvable molecular species incorporate phosphate, and the more highly-phosphorylated band migrates faster, rather than slower, than the other in acid-urea gel systems. Incorporation of [3H]lysine into HMG-E/G following release from isoleucine deprivation G1 block indicates that the protein is extensively synthesized during both the G1 and S phases of the cell cycle.

Lung hyperpermeability and changes in biochemical constituents in bronchoalveolar lavage fluids following X irradiation of the thorax.

Groups of rats were administered different doses of X rays (7.5 and 15 Gy), and the effect on the permeability of their lungs was evaluated during a time frame within which radiation pneumonitis develops. Sham-exposed animals served as controls. End points surveyed included lung weight and increases in the total protein in the lavage fluid. To obtain more detailed information about hyperpermeability and to examine some specific protein changes that occur in the lung's fluid in response to X irradiation, the lavage fluids were subjected to a reverse-phase HPLC technique that resolves 11 fractions quantitatively, including transferrin, albumin, and immunoglobulins derived from blood, as well as eight other protein and nonprotein constituents that appear to be derived from the lung (fractions 1, 2, 6-11). The earliest change following the 7.5-Gy dose was a decrease in fraction 6 at 1 week after exposure. As of Week 5, the lung weight and total protein in the lavage fluid were all normal, while the HPLC analyses revealed significant and equivalent increases in the amount of transferrin, albumin, and immunoglobulins in the lavage fluid; fraction 6 was no longer diminished. At 9 and 13 weeks, hyperpermeability could no longer be detected, while fraction 6 was again decreased at week 13. Fraction 6 was also decreased 1 week after the 15-Gy dose. At 5 weeks, when the weight of the lungs and the total protein in the lavage fluid were elevated, lavage fractions 1, 2, 10, and 11 were all increased, and transferrin, albumin, and immunoglobulins were increased approximately 1500, 1000, and 500%, respectively, and fractions 6 and 9 were decreased. By Week 7, the weight of the lungs returned to control limits, while total protein in the lavage fluid remained elevated. The hyperpermeability was characterized by increases in transferrin and albumin in the lavage fluid, but not immunoglobulins. Fractions 1, 2, 10, and 11 returned to within normal limits, whereas fraction 9 decreased further. Increases in transferrin and albumin were components of a persisting hyperpermeability observed at the last 9-week time point. All other fractions were normal, with the exception of fraction 6, which remained decreased.(ABSTRACT TRUNCATED AT 400 WORDS)

Lung injury following exposure of rats to relatively high mass concentrations of nitrogen dioxide.

Human inhalation exposures to relatively high mass concentrations of the oxidant gas nitrogen dioxide (NO2) can result in a variety of pulmonary disorders, including life-threatening pulmonary edema, pneumonia, and bronchiolitis obliterans. Inasmuch as most experimental studies to date have examined NO2-induced lung injury following exposures to near ambient or supra-ambient concentrations of NO2, e.g., < or = 50 ppm, little detailed information about the pulmonary injurious responses following the acute inhalation of higher NO2 concentrations that are more commensurate with some actual human exposure conditions is currently available. Described in this report are the results from a series of investigations in which various aspects of the inhalation toxicity of high concentrations of NO2 have been examined in laboratory rats. In the first component of our study, we characterized the kinetic course of development of lung injury following acute exposures to high concentrations of NO2 delivered over varying durations, and we assessed the relative importance of NO2 exposure concentration versus exposure time in producing lung injury. For a given exposure duration, the resulting severity of lung injury was found to generally scale proportionately with inhaled mass concentration, whereas for a given concentration of inhaled NO2, the magnitude of resulting injury was not directly proportional to exposure duration. Moreover, evidence was obtained that indicated exposure concentration is more important than exposure time when high concentrations of NO2 are inhaled. In a second component of our investigation, we assessed the pulmonary injurious response that occurs when NO2 is inhaled during very brief, 'high burst' exposures to very high concentrations of NO2. Such exposures resulted in significant lung injury, with the magnitude of such injury being directly proportional to exposure concentration. Comparisons of results obtained from this and the first component studies additionally revealed that brief exposures to the very high concentrations of NO2 are more hazardous than longer duration exposures to lower concentrations. In a third study series, we examined pre-exposure, exposure, and post-exposure modifiers of NO2-induced lung injury, including dietary taurine, minute ventilation, and post-exposure exercise. Results from these studies indicated: (i) dietary taurine does not protect the rat lung against high concentration NO2 exposure, (ii) the severity of acute lung injury in response to NO2 inhalation is increased by an increase in minute ventilation during exposure, and (iii) the performance of exercise after NO2 exposure can significantly enhance the injurious response to NO2.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of mitotic-specific histone phosphorylation by sodium arsenite.

Synchronized cultures of Chinese hamster cells (line CHO) were used to measure the effects of 10 mum sodium arsenite on histone phosphorylation. This treatment caused cell proliferation to be temporarily arrested, after which the cells spontaneously resumed cell proliferation in a radiomimetic manner. Immediately following treatment, it was found that sodium arsenite affected only mitotic-specific H1 and H3 phosphorylations. Neither interphase, nor mitotic, H2A and H4 phosphorylations were affected, nor was interphase H1 phosphorylation affected. The phosphorylation of H1 was inhibited only in mitosis, reducing H1 phosphorylation to 38.1% of control levels, which was the level of interphase H1 phosphorylation. The phosphorylation of both H3 variants was inhibited in mitosis, the less hydrophobic H3 to 19% and the more hydrophobic H3 to 24% of control levels. These results suggest that sodium arsenite may inhibit cell proliferation by interfering with the cyclin B/p34(cdc2) histone kinase activity which is thought to play a key role in regulating the cell cycle. It has been proposed that H1 and H3 phosphorylations play a role in restructuring interphase chromatin into metaphase chromosomes. Interference in this process by sodium arsenite may lead to structurally damaged chromosomes, resulting in the increased cancer risks known to be produced by arsenic exposure from the environment.

Biological availability of nickel arsenides: toxic effects of particulate Ni5As2.

To determine if fugitive nickel arsenides from an oil shale retort could pose a threat to living organisms, we studied the effects of particulate Ni5As2 on cultured mammalian cells. Culture growth rate was greatly reduced at the lowest suspension concentration tested (10 microM), even though much of the Ni5As2 powder remained insoluble. FCM analysis indicated Ni5As2 arrested cell-cycle traverse in G1 and G2. Cell survival studies indicated cells could overcome this toxicity if exposure levels were low (less than or equal to 25 microM in suspension) and restricted to less than or equal to 24 h. At higher powder levels, survival was greatly reduced. Transmission electron microscopy (TEM) demonstrated that cells exposed to less than or equal to 100 microM powder did not phagocytize the Ni5As2 particles. At higher concentrations, TEM X-ray microanalysis demonstrated that As was preferentially extracted from the Ni5As2 particle surface and free Ni was deposited inside the cell. These observations suggest that the toxicity of Ni5As2 particles may be caused by some soluble product of Ni5As2.

Analysis of nucleoproteins by direct injection of dissolved nuclei or chromosomes into a high-performance liquid chromatographic system.

This work describes the development of a method for fractionating the total nuclear proteins of Chinese hamster cells (line CHO) by high-performance liquid chromatography of whole nuclei or chromosomes. Nuclei or chromosomes were dissolved for 2 h in 6 M guanidine hydrochloride containing 0.2% trifluoroacetic acid (TFA). Residual particulates were removed by centrifugation, and a sample of the solution was passed through a Bio-Sil TSK guard column, followed by a Bio-Sil TSK 400 column, equilibrated with water containing 0.2% TFA. These columns fractionated the sample by adsorbing the DNA, passing the proteins near the exclusion limit, passing the guanidine at the inclusion limit, and passing an unidentified non-protein/non-DNA material after the inclusion limit. The protein peaks from the TSK columns were collected directly on a muBondapak CN reversed-phase column. The protein fractions were then eluted from the CN column with an acetonitrile gradient containing 0.2% TFA. Three other reversed-phase columns were examined for use with the TSK columns. The muBondapak C18 Radial-Pak column produced the best resolution of histone variants, while the Zorbax C8 column produced a better resolution of the non-histone proteins. The Nova-Pak C18 Radial-Pak column was found to be unsatisfactory for both classes of nucleoproteins.

Cell cycle kinetics of Chinese hamster (CHO) cells treated with the iron-chelating agent picolinic acid.

Spontaneously transformed (tumorigenic) Chinese hamster cells (line CHO) do not exhibit picolinic acid-sensitive G1 and G2 cell cycle arrest points observed in normal and virus-transformed cells. Rather, picolinic acid arrests CHO cells in S phase only and produces culture growth behaviour similar to that produced by hydroxyurea. Prolonged treatment with picolinic acid permits a slow but significant traverse of cells through S phase. Thus, like hydroxyurea, picolinic acid is not a useful agent for synchronizing exponential CHO cells, but it can be used to resynchronize cultures in early S phase if a previous synchronization procedure (such as isoleucine deprivation) is used. The iron chelating properties of picolinic acid, and the similarities of its effects on cultured cells to those of hydroxyurea and the iron-chelating drug desferrioxamine, suggest that picolinic acid inhibits DNA synthesis by interfering with the iron-dependent production of a stable free organic radical which is essential for the ribonucleotide reductase formation of deoxyribonucleotides.

An improved sum-of-normals technique for cell cycle distribution analysis of flow cytometric DNA histograms.

An improved method for determining the fraction of cells in the G1, S and G2 + M phases of the life cycle, from DNA distributions of S phase rich cultures, is presented. Picolinic acid synchronized cell cultures were used to demonstrate the usefulness of the technique for the cell cycle distribution analysis. The algorithm presented quantifies DNA histograms from FCM analyses using an extension of the previously reported 'sum of discrete normal curves' technique. It alleviates problems encountered with earlier methods by extending the contribution of the S phase cells to the distribution under the G1 and G2 + M peaks; at the same time it allows more of the descriptive parameters to be determined from the individual histograms. The fractions of cells in the G1, S, and G2 + M phases of the life cycle obtained by this method compare favourably with autoradiographic analyses and other analytical results.

Characterization of the mitotic specific phosphorylation site of histone H1. Absence of a consensus sequence for the p34cdc2/cyclin B kinase.

32P-Labeled histone H1 was isolated from synchronized Chinese hamster (line CHO) cells, subjected to tryptic digestion, and fractionated into 15 phosphopeptides by high performance liquid chromatography. These phosphopeptides were grouped into five classes having different cell cycle phosphorylation kinetics: 1) peptides reaching a maximum phosphorylation rate in S and then declining in G2 and M, 2) peptides reaching a maximum phosphorylation rate in G2 and then remaining constant or declining in M, 3) peptides with increasing phosphorylation throughout S and G2 and reaching a maximum in M, 4) one peptide that was phosphorylated only in M, and 5) peptides that had low levels of phosphorylation that remained constant throughout the cell cycle. Amino acid analysis and sequencing demonstrated that the mitotic specific H1 phosphopeptide was the 16-amino acid, N-terminal, tryptic peptide Ac-SETAPAAPAAAPPAEK of the H1-1 class. This peptide, which is phosphorylated on both the Ser and Thr, does not contain the consensus sequence (S/T)PXZ (where X is any amino acid and Z is a basic amino acid). This sequence is thought to be required by the p34cdc2/cyclin B kinase that has maximum phosphorylating activity in mitosis. These data indicate that this kinase either does not have an obligatory requirement for the consensus sequence in vivo as generally believed or that it is not the enzyme responsible for the mitotic specific H1 phosphorylation.

High-performance capillary electrophoresis of histones.

A high-performance capillary electrophoresis (HPCE) system was developed for the fractionation of histones. This system involves electroinjection of the sample and electrophoresis in 0.1 M phosphate buffer (pH 2.5) in a 35 cm x 50 micron I.D. coated capillary. Electrophoresis was accomplished in 9 min, separating a whole histone preparation into its components in the following order of decreasing mobility: (MHP) H3, H1 (major variant), H1 (minor variant), (LHP) H3, (MHP) H2A (major variant), (LHP) H2A, H4, H2B and (MHP) H2A (minor variant), where MHP is the more hydrophobic component and LHP is the less hydrophobic component. This order of separation is very different from that found in acid-urea polyacrylamide gel electrophoresis and in reversed-phase high-performance liquid chromatography and, thus, brings the histone biochemist a new dimension for the qualitative analysis of histone samples.

Sequential phsophorylation of histone subfractions in the Chinese hamster cell cycle.

Ion exchange chromatography and preparative electrophoresis were used to examine the phosphorylation of histone f1 and f3 subfractions in synchronized Chinese hamster cells (line CHO). Three discrete f1 phosphorylation events were demonstrated to occur in sequence during the cell cycle. The first event (f1G1) commenced in G1 2 hours prior to entry of cells into S phase; the second event (f1s) commenced simultaneously with initiation of DNA synthesis; and the third event (f1M) commenced when cells entered mitosis. F1M phosphorylation occurred simultaneously with the phosphorylation of histone f3 (which is not phosphorylated during G1, S, or G2). Fractionation of f1 and f3 revealed no differences in these sequential phosphorylation patterns among the various f1 and f3 subfractions, indicating that these phosphorylations are general biochemical events of the cell cycle. Phosphorylated (f1G1) was found to accumulate in cells as they traversed THEIR CELL CYCLE. F1s was phosphorylated to twice the extent of f1G1, but f1s did not accumulate in the cells as they passed through interphase. F1M was phosphorylated to about 4 times the extent of the first phosphorylated form (f1G1). A model of the relationship of histone phosphorylation to the cell cycle is presented which suggests that (a) f1G1 phosphorylation is involved with chromatin structural changes necessary for cell proliferation; (b) f1s phosphorylation is involved with DNA replication; (c) F1M and f3 phosphorylations are involved in chromosome condensation.

Effects of caffeine on radiation-induced phenomena associated with cell-cycle traverse of mammalian cells.

Caffeine induced a state of G(1) arrest when added to an exponentially growing culture of Chinese hamster cells (line CHO). In addition to its effect on cell-cycle traverse, caffeine ameliorated a number of the responses of cells to ionizing radiation. The duration of the division delay period following X-irradiation of caffeine-treated cells was reduced, and the magnitude of reduction was dependent on caffeine concentration. Cells irradiated during the DNA synthetic phase in the presence of caffeine were delayed less in their exit from S, measured autoradiographically, and the radiation-induced reduction of radioactive thymidine incorporation into DNA was lessened. Cells synchronized by isoleucine deprivation, while being generally less sensitive to the effects of ionizing radiation than mitotically synchronized cells, were equally responsive to the effects of caffeine. The X-ray-induced reduction of phosphorylation of lysine-rich histone F1 was less in caffeine-treated cells than in untreated cells. Finally, survival after irradiation was only slightly reduced in caffeine-treated cells. A possible role of cyclic AMP in cell-cycle traverse of irradiated cells is discussed.

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.