Preferential effect of bleomycin on newly replicated chromatin in nuclei from L1210 cells

This study determined whether nascent chromatin in nuclei from leukemia L1210 cells constitutes a preferential target for bleomycin. No differences were seen in fragmentation of nascent and bulk DNA as judged by DNA double-stranded cleavage and the release of acid-soluble material or subnucleosomal (under 8 S) fragments. In contrast, bleomycin-induced chromatin aggregation (Woynarowski, J.M., Gawron, L.S. and Beerman, T.A. (1987) Biochim. Biophys. Acta 910, 149-156) occurred preferentially in nascent chromatin as indicated by a retarded solubilization of nascent chromatin and generation of a fast-sedimenting material (above 45 S) in the sedimentation profiles of drug-released nascent chromatin. This preferential aggregation disappeared completely when chromatin became older than 10 min. The drug aggregation activity did not distinguish nascent and mature presolubilized oligonucleosomes. The results suggest that bleomycin recognizes higher-order structures of nascent chromatin.     

Conversion of 1-alkyl-2-acetyl-sn-glycerols to platelet activating factor and related phospholipids by rabbit platelets

Bleomycin is an anti-tumor agent whose cytotoxicity is related to the introduction of both single-stranded and double-stranded breaks in cellular DNA. In an assay using isolated nuclei, low levels of ethidium bromide substantially increased bleomycin induced release of nuclear chromatin. Treatment of mouse L1210 leukemia cells in vitro with low levels of ethidium bromide followed 1 hr later by bleomycin produced a synergistic effect that was 8 fold greater than that expected from the additive cytotoxicity of each drug alone. Interestingly, when the order of drug addition was reversed the drug synergism was much reduced (2 fold). The combination of DNA unwinding and strand scission agents may represent a novel and rational approach to the chemotherapy of cancer.     

Cleavage of DNA in nuclei and chromatin with staphylococcal nuclease.

Treatment of either rat liver chromatin or intact nuclei with the enzyme staphylococcal nuclease results in the conversion of about half of the DNA to acid-soluble oligonucleotides. As previously described, mild digestion of nuclei results in the liberation of a series of nucleoprotein particles containing DNA fragments which are all integral multiples of a unit length DNA 185 base pairs in length. Analysis of the kinetics of appearance of these fragments suggests that at least 85% of the nuclear DNA is involved in the formation of the repeating subunit profile. More extensive digestion of nuclei however results in the generation of a series of eight unique DNA fragments containing 160 to 50 base pairs. The series of smaller molecular weight DNA is virtually identical with the profile obtained upon limit digestion of isolated chromatin. By velocity centrifugation we have obtained highly purified preparations of the monomeric nucleoprotein particle. Digestion of this monomeric subunit results in the solubilization of 46% of the DNA and analysis of the resistant DNA again reveals the set of eight lower molecular weight fragments. These data suggest that the initial site of nuclease cleavage in chromatin resides within the DNA bridging the repeating monomeric subunits. Further attack results in cleavage at a set of sites within the monomer liberating a pattern of smaller DNA fragments which probably represents the points of intimate contact between the histones and DNA.     

Heterogeneity of chromatin fragments produced by micrococcal nuclease action.

Digestion of calf thymus chromatin with micrococcal nuclease produces a mixture of apparently well defined nucleoprotein fragments which have been partially resolved by sedimentation on linear (5-20%) sucrose gradients. Sedimentation patterns reveal a predominant peak at the 11S position, three slower components, which have not previously been reported, at the 3.4S, 5.3S and 8.6S positions, and three faster components at the 17S, 22S and 26S positions. DNA isolated from the 3S to 12S region of gradients has been resolved on polyacrylamide gels into nine to ten discrete components ranging from 47 to 156 base pairs in length. A nearly identical pattern of small DNA products was obtained from chromatin digested in intact nuclei. These data suggest that chromatin contains either several types of subunits or predominently a single type of subunit which can be asymmetrically cleaved at any one of four or more sites.     

Preparation and physical characterization of a homogeneous population of monomeric nucleosomes from HeLa cells.

We describe a method of isolating a homogeneous population of "trimmed" monomeric nucleosomes from Hela cells. These nucleoprotein particles contain a 140 +/- 5 base pair length of DNA and have a histone/DNA ratio of 1.2. They lack H1 and contain equal amounts of the four smaller histones. The DNA contains no single strand nicks. The particles sediment with an S20,w of 11S in D2O density gradients. After formaldehyde fixation, they band at a density of 1.4370 in neutral CsCl. Digestion of nucleosomes with either micrococcal nuclease or DNase I generates the same pattern of DNA fragments observed when intact nuclei are digested. Circular dichroism spectra indicate that the 280 nm positive ellipticity maximum of nucleosomes is about one-half that of chromatin. In the presence of 6 M urea, nucleosomes sediment with an S20,w of 6S, have a multiphasic thermal denaturation profile, and exhibit a circular dichroic spectrum nearly identical to that of B-form DNA. Our yield of purified nucleosomes (10-15% of the input DNA) is similar to the yields of other methods; our nucleosome population is substantially more homogeneous than those previously reported.     

Nuclease-sensitive sites in the two major intracellular simian virus 40 nucleoproteins

Simian virus 40 nucleoprotein isolated from the nuclei of infected cells contains a nuclease-sensitive site adjacent to the viral origin of replication (between 0.66 and 0.73 map unit). Nuclear extracts were subfractionated by sucrose gradient centrifugation to yield provirions (200S) and simian virus 40 chromatin (80S). The 80S fraction was cleaved either by DNase I or by an endonuclease endogenous to BSC-1 cells with high preference for the 0.66 to 0.73 region. The 200S fraction was treated to release core particles that were sensitive to nuclease cleavage; however, DNase I showed little or no preference for the 0.66 to 0.73 region of the provirion core nucleoprotein.     

Mitochondria-independent morphological and biochemical apoptotic alterations promoted by the anti-tumor agent bleomycin in Saccharomyces cerevisiae.

Bleomycin is a highly potent cytotoxic and genotoxic agent used in the chemotherapy of various types of tumors. It is a radiomimetic anticancer drug that produces single- and double-stranded DNA breaks in a catalytic way. Using Saccharomyces cerevisiae as a model system, we show that when a high amount of bleomycin molecules is internalized (100 micromol/L), morphological changes identical to those usually associated with apoptosis, i.e., a sub-G1 region peak, chromatin condensation, and very rapid DNA fragmentation into oligonucleosomal-sized fragments, are observed. The known bleomycin inhibitors cobalt and EDTA were able to prevent bleomycin nucleasic activity and thus apoptotic cell death. However, both oligomycin, a potent inhibitor of the mitochondrial F0F1-ATPase, and antimycin, a drug affecting mitochondria respiration, were unable to prevent the bleomycin-induced apoptotic-like cell death. These results suggest that high bleomycin concentrations induce an apoptosis-like mitochondria-independent cell death in yeast.     

Nucleoprotein complexes of minute virus of mice have a distinct structure different from that of chromatin.

We studied the structure of viral nucleoprotein complexes extracted from the nuclei of mouse cells infected with the immunosuppressive strain of the minute virus of mice (MVMi). Two types of complex were detected, with sedimentation coefficients of about 110 and 40S. The complexes sedimenting at 110S contained single-stranded MVMi DNA as well as a second form of viral DNA which apparently had a heat-sensitive secondary structure. The 110S peak also contained proteins which coelectrophoresed with the MVMi capsid proteins. Complexes sedimenting at 40S contained the double-stranded replicative form of MVMi DNA. These complexes sedimented faster than did the pure replicative form DNA (15S), but more slowly than cellular chromatin fragments containing DNA of the same length. They incorporated labeled deoxynucleoside triphosphate in vitro into the replicative form DNA. We investigated the structure of MVMi nucleoprotein complexes in the following ways. Nuclei of MVMi-infected cells were digested with staphylococcal nuclease, and the resulting DNA fragments were electrophoresed, transferred to nitrocellulose, and hybridized first with labeled MVMi DNA and then with cellular DNA. A nucleosomal repeat pattern was seen with the cellular DNA probe but not with the MVMi DNA probe. The DNA in MVMi nucleoprotein complexes was cross-linked with psoralen, purified, denatured, and examined with an electron microscope. Bubbles, indicating the presence of proteins, were seen in the MVMi DNA. The length of the DNA in the bubbles was 90 +/- 29 nucleotides. On the other hand, nucleosomes protected 160 base pairs from cross-linking by psoralen. The MVMi nucleoprotein complexes thus have a distinct structure which is different from that of chromatin.     

Nucleosome repeat structure is present in native salivary chromosomes of Drosophila melanogaster

The regularly repeating periodic nucleosome organization is clearly resolved in the chromatin of the isolated salivary chromosomes of Drosophila melanogaster. A new microsurgical procedure of isolation in buffer A of Hewish and Burgoyne (1973, Biochem. Biophys. Res. Commun., 52:504-510) yielded native Drosophila salivary chromosomes. These chromosomes were then swollen and spread by a modified Miller procedure, stained or shadowed, and examined in the electron microscope. Individual nucleoprotein fibers were resolved with regularly repeated nucleosomes of approximately 10 nm diameter. Micrococcal nuclease digestion of isolated salivary nuclei gave a family of DNA fragments characteristic of nucleosomes for total chromatin, 5S gene, and simple satellite (rho = 1.688 g/cm3) sequences.     

Properties of chromatin subunits from developing trout testis.

When a sample of trout testis nuclei is digested with micrococcal nuclease, the DNA is cleaved almost entirely to discrete fragments approximately 200 base pairs long and multiples thereof. The same DNA fragments can be obtained when isolated chromatin, as opposed to intact nuclei, is nuclease digested. These DNA fragments can also be found in discrete chromatin "subunits" isolated from nuclease-digested nuclei. Sedimentation through sucrose gradients or velocity sedimentation in an analytical ultracentrifuge separates these chromatin subunits into 11 S (monomer), 16 S (dimer), and 22 S (trimer) etc. species. Subunits can also be fractionated on a Sepharose 2B column equilibrated and run in low salt. High salt (greater than 40 mM NaCl) or divalent cations (congruent to 5 mM) cause subunit precipitation. Chromatin subunits have a protein to DNA ratio of approximately 1.2 and contain all the histones, including the trout-specific histone T. There are, however, no detectable nonhistone chromosomal proteins. Mg-2+ precipitates of the 11 S chromatin monomers, when pelleted, are thin and clear, while oligomer Mg-2+ pellets are thick and white. This could reflect a more symmetrical or ordered packing of 11 S monomers, which are deficient in histone I. This histone may cross-link the larger oligomers, resulting in a disordered Mg-2+ complex. These results are consistent with the subunit model of chromatin structure, based on 200 base pair long regions of DNA associated with histones. These subunits would be separated by nuclease-sensitive DNA spacer regions and cross-linked by histone I.     

Comparative study of the condensation of chicken erythrocyte and calf thymus chromatins by di- and multivalent cations

The condensation of chicken erythrocyte (CE) and calf thymus (CT) chromatins upon addition of di- and multivalent cations has been studied using turbidity, precipitation and electric dichroism measurements. For all the cations investigated (Mg2+, Tb3+, Co(NH3)6(3+), spermidine Spd2+ and spermine Sp4+) condensation of CE chromatin occurred before the onset of aggregation, while aggregation of CT chromatin started before condensation with all cations except Mg2+ and Tb3+. Precipitation of CE chromatin required lower di- and multivalent cations concentrations than CT chromatin. The electric dichroism data for both chromatins, at low ionic strength in the absence of di- or multivalent cations, indicated that the nucleoprotein molecules were not totally decondensed but that a "precondensed" state was already present. A positive electric dichroism was observed for the most condensed chromatin fibers, in agreement with the "cross-linker" models. Tb3+ led to less compact condensed particles as judged from the electric dichroism observations, but electron microscopy revealed that "30 nm fibers" were formed. Very little aggregation was produced by Tb3+. On the contrary, spermine produced very large networks of condensed molecules, but large spheroidal particles were also observed. The condensation of CE chromatin happened without changes of solution conductivity upon cation salt addition, regardless of the condensing cation, indicating a cooperative uptake of the ions during this process.     

Effect of the antitumor antibiotic bleomycin on DNA polymerase activity

Treatment with bleomycin activates considerably a repair synthesis of DNA in rat liver chromatin in vitro and can cause loosening of the nucleoprotein complex, which facilitates the accessibility or repair enzymes for lesions in chromatin DNA. The bleomycin action on DNA-template increases severalfold the rate of synthesis catalyzed by DNA polymerase beta inhibits the activity of DNA polymerase I from Escherichia coli and suppresses severalfold the activity of DNA polymerase alpha and DNA polymerase of bacteriophage T4. The effect of bleomycin consists in a prevailing increase of nicks and minimal gaps in DNA as compared to the rise of moderate gaps, thus suggesting that bleomycin is a gamma-mimetic.     

Accessibility of some regions of DNA in chromatin (chicken erythrocytes) to single strand-specific nucleases.

The susceptibility of the DNA in chromatin to single strand-specific nucleases was examined using nuclease P1, mung bean nuclease, and venom phosphodiesterase. A stage in the reaction exists where the size range of the solubilized products is similar for each of the three nucleases and is nearly independent of incubation time. During this stage, the chromatin fragments sediment in the range of 30 to 100 S and contain duplex DNA ranging from 1 to 10 million daltons. Starting with chromatin depleted of histones H1 and H5 similar fragments are generated. In both cases these nucleoprotein fragments are reduced to nucleosomes and their multimers by micrococcal nuclease. Thus, chromatin contains a limited number of DNA sites which are susceptible to single strand-specific nucleases. These sites occur at intervals of 8 to 80 nucleosomes and are distributed throughout the chromatin. Nucleosome monomers, dimers, or trimers were not observed at any stage of single strand-specific nuclease digestion of nuclei, H1- and H5-depleted chromatin, or micrococcal nuclease-generated oligonucleosomes. Each of the three nucleases converted mononucleosomes (approximately 160 base pairs) to nucleosome cores (approximately 140 base pairs) probably by exonucleolytic action that was facilitated by the prior removal of H1 and H5. The minichromosome of SV40 is highly resistant to digestion by nuclease P1.     

Biochemical and cytological studies of bleomycin actions on chromatin and chromosomes

Interaction of bleomycin with nuclei isolated from a variety of mammalian cells resulted in the release of nucleosomes. When isolated mononucleosomes (core plus linker) were re-treated with bleomycin, no further degradation of DNA occurred. The results suggest that the bleomycin cleavage sites in chromatin are present only in the linker region and that there are probably only one or two cleavage sites per linker. The repeat lengths of nucleosomal DNA released by bleomycin from nuclei of different species are different; this variability is considered to reflect the length of the linker. Incorporation of BrdU into DNA did not alter the bleomycin action on nucleosomes. When mitotic cells were held at metaphase for a prolonged period, bleomycin caused a gradual disintegration of chromosomes, although the bleomycin cleavage sites in metaphase chromosomes were found to be the same as those in interphase nuclei.     

Scatter analysis of discrete-sized chromatin fragments favours a cylindrical organization

Fragments of chromatin containing 23 +/- 2.5 nucleosomes have been fractionated after light nuclease treatment of chicken erythrocyte nuclei. Low-angle scattering measures the total z-average radius of gyration of the already well-defined particles and the shape of scatter curves can be compared with three-dimensional analysis as opposed to cross-section analysis of long chromatin fragments. The data show that the particles are not spherical, have no detectable hole in the center of the structure and are best represented by a solid rod-like shape such as that generated by a coil of nucleosomes with the centre perhaps filled with linker DNA and histone H1/H5. 23 nucleosome fragments, where the DNA is partially fragmented, have near-identical scatter curves to the above-defined intact particles, indicating the primary importance of histone proteins in maintaining the integrity of the chromatin higher-order structure. Neutron scattering shows the radii of gyration to be contrast-independent, which fits in with the model calculations for solenoids. Particles with fragmented DNA and the intact particles, therefore, behave as sections of a solenoidal higher-order structure and possibly are observed as "superbeads' only during the folding and unfolding pathways of nucleosome multimers.     

Structure of chromatin and chromosomes in preparations of interphase nucleus derivatives, prepared by removal of the nucleuar envelopes. II. Structure of chromatin and associations of chromosomes in stretched amembranous nuclei and mitotic figures]

Preparations of surface stretched amembranous nuclei and mitotic figures were used for revealing the high order nuclear and chromosomal structures. The preparations were obtained by dropping amembraneous nuclei and mitotic figures suspension in methanol-glacial acetic acid mixture (3:1) on wetted superclean slides. Amembraneous nuclei and mitotic figures were isolated from intact murine and human cells (lines L1210, SK-UT-1B, PHA-stimulated lymphocytes) by means of their 1-5 min prefixational capillary pipetting with freshly prepared 0.018-0.06% Triton X-100 solution in the conditional cultural medium. Stretched amembraneous nuclei and mitotic figures had no features of induced chromatin dispersion and compaction. Stretched interphase amembraneous nuclei showed spatially separated individual structures (thin chromatin fibres, nucleoli, intranuclear bodies), polymorphous pattern of perinucleolar chromatin aggregation and episodically expressed beaded thick chromatin fibres and a chromocenter. The chromomeric pattern of the spread chromosomes of mitotic figures was quite similar but hardly identical with that of G-banding. The stretched prometaphase mitotic figures in all tested cell types always contained loose "residual" nucleoli looking like typical prophase nucleoli as concerns their shape and number per cell (mitotic figure). The majority of chromosomes of stretched mitotic figures and of prophase amembraneous nuclei were attached to the nucleolar material. All tested cell lines showed almost the same variation in number of nucleolus-attached chromosomes, per both prophase amembraneous nucleus and prometaphase mitotic figure. Some chromosomes of stretched mitotic figures were colocated with "residual" nucleoli and looked shortened and strongly condensed. Other chromosomes, locally associated with "residual" nucleoli, were straight and oriented radially to these. Mutual chromosomal arrangements in mitotic cells on smears and in stretched mitotic figures were analogous. Equatorial plates from PBS-washed SK-UT-1B cells displayed a better stretching capacity than those from untreated cells. In the former case metaphase chromosomes were seen more uniformly stretched and well identified after GTG-banding procedure. The number of interchromosomal (mainly telomere-telomeric and telomere-centromeric) connections per stretched mitotic figure (or per stretched prophase amembraneous nucleus) was minimum in late prometaphase, maximum in prophase and early prometaphase, and intermediate in metaphase. The obtained data are discussed in terms of topology and longitudinal heterogeneity of mitotic chromosomes.     

Degradation of HeLa cell chromatin by neocarzinostatin and its chromophore

Chromatin is the in vivo target site for neocarzinostatin, a DNA strand scission antitumor drug. The effect of neocarzinostatin and its active chromophore component on HeLa cell chromatin is described here. Chromatin consisting of a mixture of mono-, di-, tri- and larger nucleosome fragments is prepared by micrococcal nuclease digestion of HeLa cell nuclei. Drug-induced conversion of chromatin to smaller sized fragments is measured by electrophoresis of the DNA on non-denaturing 4% polyacrylamide gels. Chromatin breakdown measured under these conditions is double-stranded in nature. In the presence of 2 mM dithiothreitol, neocarzinostatin causes degradation of large chromatin fragments and a loss of distinct nucleosome peaks. Detection of chromatin breakdown by neocarzinostatin is dependent upon the concentration of chromatin in the assay. When chromatin is increased from 14 to 70 micrograms/ml, changes in the larger fragments caused by 100 micrograms/ml neocarzinostatin become less obvious are are almost undetectable at 140 micrograms/ml chromatin. No change is observed when chromatin is treated with either neocarzinostatin or its chromophore in the absence of dithiothreitol. For detectable levels of chromatin degradation, 10 micrograms/ml neocarzinostatin is required compared to only 2.5 microgram/ml chromosome (expressed in microgram equivalent neocarzinostatin). Such degradation also occurs more rapidly with chromophore than with neocarzinostatin. Digestion of chromatin with neocarzinostatin continues for at least 30 min at 37 degrees C, while similar degradation caused by chromophore is complete in 1 min. Neocarzinostatin levels which actively degrade isolated chromatin can also effect release of soluble chromatin from intact nuclei. The released chromatin can serve as a substrate for micrococcal nuclease digestion. Such chromatin studies should prove useful in characterizing the mechanism of action of DNA reactive drugs such as neocarzinostatin.