Effects of hyperthermia, irradiation, and cytotoxic drugs on fluorescein isothiocyanate staining intensity for flow cytofluorometry

Measurement of fluorescein isothiocyanate (FITC) staining intensity of cultured lymphoblastoid cells following hyperthermia showed large increases without concomitant increases in nuclear protein. Similar measurements of cells following incubation with cytotoxic drugs showed fluorescent intensity increases that exceeded the increases in nuclear protein that were due to the cell cycle blocking action of the drug. The reverse, however, was true for cells following irradiation. In contrast, FITC staining intensity and nuclear protein measurements of cells proceeding through the cell cycle after removal of the cycle blocking agent showed nearly parallel changes, although there were reproducible minor differences, especially following blocking with hydroxyurea. These results suggest that FITC staining intensity is a function not only of nuclear protein content but also of stain access to the reaction sites of the protein constituents of the chromatin. Thus, it is possible that FITC staining may be used as a probe of changes in chromatin structure following experimental manipulation of cells in vitro or treatment of tumors in vivo.     

Programmed Cell Death in Plants: Ultrastructural Changes in Pea Guard Cells

Treatment with cyanide of epidermal peels isolated from pea leaves resulted in destruction of nuclei in the guard cells of stomata, which is visible with a light microscope. The process was accelerated by illumination. Electron microscopy revealed significant CN--induced changes in the ultrastructure of guard cells, which increased with time. Margination of chromatin, which is one of the first signs of apoptosis, was observed in the guard cells even after 1 h incubation of the isolated epidermis with CN-. Subsequent chromatin condensation, swelling of the endoplasmic reticulum with formation of large tanks covered with ribosomes, changes in the structure of dictyosomes, and a slight swelling of mitochondria were observed after 3 h of the epidermis incubation with CN-. After 6 h of incubation with CN-, the bulk volume of the guard cells was filled with vacuoles, the cytoplasm occupied the thin marginal layer, the nucleus was in the center similarly to the control experiment, but it was polylobal, extended in narrow cytoplasmic bands, and, despite the loss of the nuclear envelope integrity, appeared to be a self-dependent structure. In the envelope-free open regions of the nucleus, mitochondria and chloroplasts directly contacted with chromatin. Much like the cell nucleus, chloroplasts lost the integrity of the membrane, but did not swell and retained the stroma and integrity of the thylakoid system. An antioxidant di-tert-butyl-4-hydroxytoluene prevented ultrastructural changes in the cells observed after 6 h of incubation with CN-. Thus, the CN--induced death of the guard cells of stomata occurs through the mechanism of apoptosis.     

Sequence-specific potentiation of topoisomerase II inhibitors by the histone deacetylase inhibitor suberoylanilide hydroxamic acid

Acetylation of histones leads to conformational changes of DNA. We have previously shown that the histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), induced cell cycle arrest, differentiation, and apoptosis. In addition to their antitumor effects as single agents, HDAC inhibitors may cause conformational changes in the chromatin, rendering the DNA more vulnerable to DNA damaging agents. We examined the effects of SAHA on cell death induced by topo II inhibitors in breast cancer cell lines. Topo II inhibitors stabilize the topo II-DNA complex, resulting in DNA damage. Treatment of cells with SAHA promoted chromatin decondensation associated with increased nuclear concentration and DNA binding of the topo II inhibitor and subsequent potentiation of DNA damage. While SAHA-induced histone hyperacetylation occurred as early as 4 h, chromatin decondensation was most profound at 48 h. SAHA-induced potentiation of topo II inhibitors was sequence-specific. Pre-exposure of cells to SAHA for 48 h was synergistic, whereas shorter pre-exposure periods abrogated synergy and exposure of cells to SAHA after the topo II inhibitor resulted in antagonistic effects. Synergy was not observed in cells with depleted topo II levels. These effects were not limited to specific types of topo II inhibitors. We propose that SAHA significantly potentiates the DNA damage induced by topo II inhibitors; however, synergy is dependent on the sequence of drug administration and the expression of the target. These findings may impact the clinical development of combining HDAC inhibitors with DNA damaging agents.     

In vitro interaction of 63-nickel(II) with chromatin and DNA from rat kidney and liver nuclei

The interaction of nickel(II) with chromatin was studied in vitro and in isolated nuclei from rat liver and kidney. Nickel(II) bound to chromatin, polynucleosomes (DNA + histone octamer protein complex), and to deproteinized DNA both in intact nuclei and in vitro. The amount of nickel(II) bound depended on the concentration of nickel(II), the presence of chromosomal proteins and the binding sites on DNA which provide a stable coordination environment for nickel(II). The binding of nickel(II) to chromatin and to DNA in whole nuclei was much slower than in vitro indicating that assessibility of the DNA binding sites was influenced by the presence of the nuclear membrane, nuclear matrix and nuclear proteins and/or by the condensed nuclear structure of chromatin. Since DNA containing bound nickel(II) was isolated from chromatin, nickel(II) directly interacted with stable binding sites on the DNA molecule in chromatin. Nickel(II) was associated with the histone and non-histone nuclear proteins as well as the DNA in rat liver and kidney chromatin. Nickel(II) was found to bind to calf thymus histones in vitro. Nickel(II)-nuclear protein and -DNA interactions were investigated by gel electrophoretic analysis of in vitro incubation products. Although nickel-histone and nickel-non-histone protein interactions were completely disrupted by the electrophoretic conditions, fluorography revealed the presence of inert nickel(II)-DNA and/or nickel(II)-DNA-protein complexes.     

Spectral imaging of red blood cells in experimental anemia of Cyprinus carpio

In the present work we have studied the effect of experimental anemia induced at both low and optimal temperatures on erythropoiesis in Cyprinus carpio. The results showed that hemoglobin concentration per cell was similar in both temperature conditions, however, red blood cell (RBC) concentration was higher at the optimal temperature. Induced anemia caused an abrupt decrease in RBC concentration, while the hemoglobin concentration per cell remained unchanged. Recovery, as shown by electron microscopy, was characterized by the release of differentiating young and intermediate cells to the peripheral blood. It was revealed that with the progression of differentiation the nucleus/cytoplasm ratio decreases, the chromatin condenses and the shape of the nucleus changes from round to elliptical. Spectral imaging revealed an increase in the optical density of chromatin with the maturation of the cells. The chromatin that was dispersed over the nuclear volume in the young cells becomes highly ordered in the mature cells. Spectral similarity mapping revealed the formation of a novel structure of high symmetry, representing chromatin rearrangement during the process of cellular differentiation.     

Early changes in liver chromatin in response to partial hepatectomy]

Guinea-pig and mouse liver chromatin responds to the partial hepatectomy by an increase in binding of a basic dye acridine orange (AO) and by a decrease of its stability to heat in thermal denaturation test in situ. Degree of the changes in AO chromatin binding is identical in the cells of different ploidy and proportional to their DNA content. Treatment of the preparations by 0.6 M NaCl solutions under conditions bringing about the selective removal of histone H1 from the cells produces in vitro changes in DNA properties taking place in cells in vivo in the course of their activation. The treatment of cells with 0.35 M NaCl solution results in the disappearance of changes occurring in the chromatin of activated cells whereas the properties of control cells remain unchanged. The data obtained are interpreted as a result of the removal of some non-histone regulatory proteins from the chromatin of activated cells that is accompanied by changes in the character of DNA-histone interaction. At the time of maximum increase of AO binding a significant intensification of endogenous RNA polymerase activity was found, the incorporation of [3H] UTP in the nucleolus being higher than that in the extranucleolar part of the nucleus. High ionic strength in the incubation medium (0.4 M (NH4)2SO4) results in drastic increase of radioactive label in the nucleus and in the disappearance of differences between activated and non-activated chromatin. It is concluded that the intensification of RNA synthesis under the influence of proliferative stimulus is more likely dependent on the additional opening of DNA-matrix than on the direct activation of the enzyme.     

Perforin-dependent nuclear targeting of granzymes: A central role in the nuclear events of granule-exocytosis-mediated apoptosis?

Programmed cell death, apoptosis, involves very distinctive changes within the target cell nucleus, including margination of the chromatin, DNA fragmentation and breakdown of the nuclear envelope. Cytolytic granule-mediated target cell apoptosis is effected, in part, through synergistic action of the membrane-acting protein perforin and serine proteases, such as granzymes A or B. Recent work using confocal laser scanning microscopy as well as other techniques supports the idea that perforin-dependent translocation of granzymes to the nucleus of target cells plays a central role in effecting the nuclear changes associated with apoptosis. In vitro experiments indicate that granzyme nuclear import follows a novel pathway, being independent of ATP, not inhibitable by non-hydrolysable GTP analogues and involving binding within the nucleus, unlike conventional signal- dependent nuclear protein import. In intact cells, perforin-dependent nuclear entry of granzymes precedes the nuclear events of apoptosis such as DNA fragmentation and nuclear envelope breakdown; prevention of granzyme nuclear translocation through bcl2 overexpression or treatment of target cells with inhibitors of caspase activation blocks these events. Nuclear localization of granzymes thus appears to be central to induction of the nuclear changes associated with cytolytic granule-mediated apoptosis.     

Fine structural alterations in Trypanosoma rhodesiense grown in vitro, treated with WR 163577

Fine-structural alterations in Trypanosoma rhodesiense trypomastigotes exposed to WR 163577, a prophylactic agent against animal African trypanosomiasis, were determined from cells grown in vitro. Exposure of trypomastigotes to a low concentration of drug resulted only in condensation of kinetoplast DNA fibrils. Exposure to higher drug concentrations caused clumping of nuclear chromatin and of cytoplasmic contents. Although alteration of kinetoplast DNA is the first detectable drug-induced change, the function of the kinetoplast in mammalian forms of African trypanosomes is unclear, and the secondary changes in the nucleus and cytoplasm may constitute the functionally significant alterations caused by WR 163577.     

Transport of gamma-interferon into the cell nucleus may be mediated by nuclear membrane receptors

Purified mouse interferon gamma (MuIFN-gamma), a lymphokine having potent antiviral, immunomodulatory, and growth inhibitory activities, is internalized (t1/2 less than 1.0 min) by mouse L929 fibroblasts via receptor-mediated endocytosis. Individual MuIFN-gamma molecules, identified by a postembedding immuno-gold technique, are then transported to the cell nucleus, perhaps through nuclear pores, into areas of dense chromatin. Purified, isolated nuclei of L929 cells bind radiolabeled MuIFN-gamma specifically and with high affinity (Kd = 2 X 10(-10) M). These nuclear membrane receptors, distinct from those for MuIFN-beta, number about 24,000/nucleus. Treatment of nuclei with trypsin prevents binding of MuIFN-gamma. The demonstration of rapid cellular uptake and transport of MuIFN-gamma into the dense chromatin, perhaps facilitated by nuclear receptors, suggests that IFN-gamma molecules, alone or bound to receptor, may directly affect genome regulation.     

Lamins A and C bind and assemble at the surface of mitotic chromosomes

To study a possible interaction of nuclear lamins with chromatin, we examined assembly of lamins A and C at mitotic chromosome surfaces in vitro. When a postmicrosomal supernatant of metaphase CHO cells containing disassembled lamins A and C is incubated with chromosomes isolated from mitotic Chinese hamster ovary cells, lamins A and C undergo dephosphorylation and uniformly coat the chromosome surfaces. Furthermore, when purified rat liver lamins A and C are dialyzed with mitotic chromosomes into a buffer of physiological ionic strength and pH, lamins A and C coat chromosomes in a similar fashion. In both cases a lamin-containing supramolecular structure is formed that remains intact when the chromatin is removed by digestion with micrococcal nuclease and extraction with 0.5 M KCl. Lamins associate with chromosomes at concentrations approximately eightfold lower than the critical concentration at which they self-assemble into insoluble structures in the absence of chromosomes, indicating that chromosome surfaces contain binding sites that promote lamin assembly. These binding sites are destroyed by brief treatment of chromosomes with trypsin or micrococcal nuclease. Together, these data suggest the existence of a specific lamin-chromatin interaction in cells that may be important for nuclear envelope reassembly and interphase chromosome structure.     

Localization of estrogen receptors in uterine cells. An appraisal of translocation.

The nuclear localization of estrogen receptors has been examined under conditions which minimize redistribution and localization artifacts. A procedure is presented which rapidly lyses suspensions of cells from immature rat uteri by using 0.04% Triton X-100 in isotonic buffer. The ‘nuclei’ which are obtained after lysis have a median diameter of 1μm and are devoid of nuclear membranes. There is close agreement between the number of cells before lysis and the number of nuclear particles after lysis. Triton X-100 gave no interference with quantitative binding of estradiol to receptor and no alteration in the sedimentation behavior of receptor on sucrose gradients containing high or low salt. Using this procedure to monitor the dynamics of estrogen receptor distribution within uterine cells after exposure to estradiol, translocation of estrogen receptor to the nucleus was observed to occur at a rate slightly slower than the rate at which estradiol was specifically bound to free cells or receptors. The difference in these rates is compatible with a model in which estradiol must first bind to the receptor before the binding complex moves to the nucleus. The rate of nuclear translocation was temperature-dependent and was observed to occur at 0 °C, provided that enough time was allowed for steroid entry, receptor charging and transit to the nucleus. Two distinct phases were observed to characterize nuclear receptor localization. In the first phase after hormone exposure, estrogen receptor progressively accumulated in the nucleus; afterwards, estrogen receptor was progressively lost from the nucleus but could not be detected in other subcellular compartments in a form still binding hormone. Since high cell viability was maintained during these manipulations, loss of nuclear receptor was not due to cell damage during in vitro incubation. These studies suggest that this decline in nuclear receptor level after hormone interaction, which is known to occur in vivo, may be a normal event during estrogen interaction with target cells.     

Nuclear texture parameters as discriminant factors in cell cycle and drug sensitivity studies

The authors have recently shown that cell cycle characteristics of in situ cell populations can be determined using the SAMBA 200 cell image processor by computing 15 densitometric and texture parameters on each Feulgen-stained nucleus and multiparametric analysis of data. The present paper displays the importance of chromatin pattern assessment and detection of conformational changes in DNA structure, based on nine nuclear texture parameters measured from the grey level cooccurrence and the run-length section matrices. Reference files were constructed by merging respective reference files (G0/G1, S, G2 and M) of MDA AG and MCF-7, two mammary epithelial cell lines presenting different morphological aspects and hormone responses, these files were found to be valid in the reclassification of any mammary epithelial cell in culture with a diploid or near diploid pattern. Moreover, the authors demonstrate that chromatin texture changes, following direct interaction of chemotherapeutic drugs with DNA, may be assessed owing to nuclear texture parameters. Consecutive to daunomycin addition (0.5 microgram/ml) and concomitant to the appearance of nuclear morphological alterations in MDA AG sensitive cells as viewed by microscopic observation, discriminant factorial analysis showed progressively increasing erroneous reclassification from 15 to 72 h of treatment. These experimental results were exploited with a kinetic mathematical model to quantify the daunomycin blocking effect: 20% in S phase and 80% in G2 phase. Interestingly, no textural change was observed on MDA A1 anthracycline resistant cells, indicating that these texture parameters may permit distinction of drug sensitive cells. This methodology 1) can be applied to test in vitro resistance-reversal molecules, 2) may be extended to other therapeutic agents giving rise to conformational changes in DNA structure, and 3) can be applied to cytopunctions or imprints of tumor biopsies with diploid-like DNA content to follow evolution of drug sensitivity or resistance during course of therapy.     

Fine Structural Alterations in Trypanosoma rhodesiense Grown In Vitro,Treated with WR 1635771

Fine-structural alterations in Trypanosoma rhodesiense trypomastigotes exposed to WR 163577, a prophylactic agent against animal African trypanosomiasis, were determined from cells grown in vitro. Exposure of trypomastigotes to a low concentration of drug resulted only in condensation of kinetoplast DNA fibrils. Exposure to higher drug concentrations caused clumping of nuclear chromatin and of cytoplasmic contents. Although alteration of kinetoplast DNA is the first detectable drug-induced change, the function of the kinetoplast in mammalian forms of African trypanosomes is unclear, and the secondary changes in the nucleus and cytoplasm may constitute the functionally significant alterations caused by WR 163577.     

Changes in arachidonic acid content of X-irradiated human lymphocytes and plasma membrane fractions

The 14C-arachidonic acid content of pre-labelled human lymphocytes was followed during in vitro incubation in untreated and X-irradiated cells. Loss of about 35-40 per cent of the incorporated radioactivity was found already at 10 minutes after irradiation as detected in whole cells, phospholipid, free fatty acid and phosphatidylcholine fractions of plasma membranes. When pulse labeling with 14C-arachidonic acid was performed at various intervals after in vitro irradiation, the uptake of precursor by irradiated cells did not differ from untreated ones. At the same time the activity of phospholipid, free fatty acid and phosphatidylcholine fractions was approximately 50 per cent lower compared to the control, then it increased gradually reaching the control level.     

Morphometric analysis of the toxic effect of ethanol on the structure of chromatin in interphase nuclei of rat hepatocytes

A method of automated morphometric analysis of hepatic cell interphase nuclear chromatin (CHIN) structure was elaborated using scanning microspectrophotometer SMP-05 ("Opton", FRG). Liver sections of rats were used to investigate hepatic cell CHIN structure. The animals were administered ethanol intraperitoneally once per day within a month period in doses 4, 6, 8, 10 g/kg (1/3-4/5 LD50). Morphometric indexes indicating dose-dependent changes in hepatic cell CHIN structure are demonstrated. Statistical analysis of morphometric data has led to the conclusion that hepatic cell CHIN structural modification depends on two factors: ethanol damaging effect and hepatic cell regeneration.     

Effect of vitamin E on transcription in isolated nuclei and rat liver chromatin in normal status and in E-hypovitaminosis]

The effect of alpha-tocopherol on the RNA-polymerase activity in isolated rat nuclei and chromatin from normal and E-deficient rats and the possible role of tocopherol-binding proteins in this process were studied. Some differences in the RNA-polymerase activities of the nuclei were found; however, in vitro added alpha-tocopherol had no effect on the level of the label incorporation into RNA. No effect of alpha-tocopherol on this process was observed after addition of cytosol either. Analysis of chromatins from normal and E-deficient rats revealed no differences in their RNA-polymerase activities. In vitro added alpha-tocopherol increased the RNA-polymerase activity of normal (but not of vitamin E-deficient) rats. Some differences in the RNA-polymerase activities were noted after addition to the incubation medium of the Triton X-100-solubilized nuclear fraction specifically binding alpha-tocopherol. This effect was enhanced in the presence of exogenous alpha-tocopherol. The susceptibility of chromatin from normal and E-deficient rats to DNAse I hydrolysis was also found to be different. It was concluded that vitamin E can influence the RNA-polymerase activity of the nuclei and chromatin as well as the chromatin structure and that alpha-tocopherol-binding proteins are necessary for the vitamin E effect on the RNA-polymerase activity to be manifested.     

DNA methylation and histone acetylation patterns in cultured bovine fibroblasts for nuclear transfer

Evidence indicates that failure of nuclear transfer (NT) embryos to develop normally can be attributed, at least partially, to the use of a differentiated cell nucleus as the donor karyoplast. It has been hypothesized that blastocyst production and development to term of cloned embryos may differ between population doublings (PDs) of the same cell line as a consequence of changes in DNA methylation and histone acetylation patterns during in vitro culture. The objective of this study was to determine gene expression patterns of the chromatin remodeling proteins DNA methyltransferase-1 (Dnmt1), methyl CpG binding protein-2 (MeCP2), and histone deacetyltransferse-1 (HDAC1), in addition, to measuring levels of DNA methylation and histone acetylation of bovine fibroblast cells at different PDs. Bovine fibroblast cell lines were established from four 50-day fetuses. Relative levels of Dnmt1, MeCP2, HDAC1, methylated DNA, and acetylated histone were analyzed at PDs 2, 7, 15, 30, 45, and 70. RNA levels of Dnmt1, HDAC1, and MeCP2 were examined using Q-PCR. Global levels of methylated DNA and acetylated histone were determined by incubation of fixed cells with an anti-5-methylcytidine and anti-acetyl-histone H3 antibody, respectively. Cells were labeled with a second antibody, counter-stained with propidium iodide and analyzed by flow cytometry. These data demonstrate that chromatin remodeling protein mRNAs involved in epigenetic modifications are altered during in vitro culture. Methylated DNA and acetylated histone patterns of in vitro cells change with time in culture. Subsequent use of these cells for NT will provide insight as to how these epigenetic modifications affect reprogramming.     

Photodynamic effects of Photofrin II on cell division in human NHIK 3025 cells

Human cervix carcinoma cells of the line NHIK 3025 were exposed to light after 18 h incubation with Photofrin II. After this photodynamic treatment cells in the interphase were retarded with respect to entry into mitosis for a period which increased with increasing light dose. Following the prolonged interphase, an increase in the mitotic index was observed, giving rise to a 3-fold higher level of mitotic cells compared to the control level. Staining of methanol-fixed cells with the DNA-specific dye mithramycin indicated that the increase in mitotic index was due to a prolongation of the metaphase. For all the light doses studied most of the metaphase cells could be characterized as three-group metaphases or c-metaphase-like structures for the first 8 h after treatment. An approximately 10-fold increase above the control level in the number of tripolar mitoses was also observed. A 2h incubation in a Photofrin II-free medium after the 18 h incubation with Photofrin II and before light exposure reduced the fluorescence of the cells by 30 per cent. However, this wash-out period had no effect on the increase in mitotic index after light exposure. A light dose corresponding to 80 per cent survival (as assayed on asynchronous cells) was given to cells in mitosis after Photofrin II incubation. This treatment delayed more than 90 per cent of the metaphase cells from entering the anaphase for at least 1 h. Cells photodynamically treated in the anaphase and telophase entered the interphase at a similar rate as control cells. These observations indicate a temporary block in the initiation of the anaphase and a prolongation of the metaphase. A microscopic study of cells immunologically stained for beta-tubulin 1 h after photodynamic treatment indicated that the organization of the spindle apparatus was disturbed by the photodynamic treatment. Such perturbations are suggested to be the cause of the observed accumulation of cells in mitosis.