S-phase Nuclei Research Articles

Effect of magnesium on the growth and cell cycle of transformed and non-transformed epithelial rat liver cells in vitro

The effects of magnesium (Mg) restriction on cell growth and the cell cycle were determined in transformed (TRL-8) and non-transformed (TRL-12-15) epithelial-like rat liver cells. Cells were cultured in RPMI 1640 medium in which the Mg concentration was reduced to 0.5, 0.1, and 0 x the concentration in the regular RPMI 1640 media (100mg/l). Cell growth in the transformed cells was not influenced by the Mg restriction as greatly as in the non-transformed cell line. Transit through the cell cycle also exhibited an independence of the Mg in the medium in the transformed cells. When transformed cells were grown for two generations in Mg-limited medium, the growth rate slowed to a rate similar to that demonstrated by the non-transformed cells. Analysis by flow cytometry showed that transit through the cell cycle was minimally slowed in Mg deficient transformed cells; however, transit through the G1 and S phases in the non-transformed cells was slowed. The TRL-8 cells in Mg-limited medium resulted in fewer nuclei in G1 with subsequent increases in the percentages of S-phase nuclei. The TRL 12-15 cells reacted oppositely with the number of G1 nuclei increased and the number of S-phase nuclei decreased. In respect to growth, these results show that epithelial cells respond in a similar manner to Mg-limitation as do fibroblast cells. The transformed cells exhibited a level of independence from Mg in respect to growth, reproduction, and cell-cycle kinetics.

Cell kinetics of regenerating liver after 70% hepatectomy in rats--2-color flow cytometric analysis.

Two-color flow cytometric (FCM) analysis using anti-bromodeoxyuridine (BrdU) monoclonal antibody (MoAb) was used to investigate the cell kinetics of regenerating liver after 70% partial hepatectomy in rats. Three peaks were seen in DNA histograms of rat hepatocyte nuclei, corresponding to diploid(2c), tetraploid(4c), and octaploid(8c). These proportions changed in the course of regeneration which were clearly demonstrated by DNA histograms using flow cytometry. The proportion of diploid, tetraploid, and octaploid nuclei in control liver were 49.3 ± 1.6%, 45.0 ± 7.4%, and 1.7 ± 0.7%, respectively. A significant change occurred at 24 hours after hepatectomy, as FCM revealed 25.9 ± 1.1% diploid, 54.5 ± 1.2% tetraploid, and 9.0 ± 0.9% octaploid. This shift to polyploid nuclei persisted until 72 hours, and then gradually returned to the pattern of control liver. The S-phase nuclei which incorporated BrdU increased rapidly at 24 hours to a peak of 11.3 ± 0.9%, and gradually decreased to 5.8 ± 0.8%, 5.3 ± 0.8%, 2.4 ± 0.6%, 2.9 ± 1.1%, and 1.2 ± 0.6%, at 48, 72, 96, 120, and 168 hours, respectively. This 2-color FCM analysis made a detailed analysis of the cell kinetics in regenerating hepatocytes possible, and may be applied in investigations of various aspects of liver regeneration.

The First Cell Division Cycle in Nicotiana Mesophyll Protoplasts Cultured in Vitro. I. Methods to Determine Cycle Kinetics

Summary The progress of freshly isolated and in vitro cultured mesophyll protoplasts through the first mitotic cycle was studied with the aim of determining the frequency and timing of the G2-phase. Putative G1 and G2 phase cells were identified by measuring the relative DNA content through improved cytofluorimetry of DAPI-stained nuclei. S-phase nuclei were identified by labelling with the thymidine-analogue BrdU, which was subsequently immuno-localized with a BrdU-specific monoclonal antibody. The cells entered S-phase after 12–24h and a maximum of G2 cells was observed 24–36h after culture initiation. Most of the cells had divided after 48 to 72 h.

The S-phase cytotoxicity of camptothecin

The DNA topoisomerase I inhibitor camptothecin (CAM) is selectively cytotoxic to S-phase cells of HL-60, and some other myelogenous leukemic lines. The early effects of cell exposure to 0.05–0.2 μg/ml CAM are seen after 2 h; at that time a progressive degradation of DNA in the chromatin of S-phase cells is initiated. The degradation manifests by “pulverization” of chromatin followed by coalescence of the fine granules and nuclear disintegration. Between 2 and 6 h of treatment, a loss of about 30–70% of DNA from S-phase nuclei is detected by flow cytometry. A 10-min pulse of CAM is adequate to trigger subsequent DNA degradation. Agarose gel electrophoresis of DNA from CAM-treated cells reveals a typical nucleosome core particles “ladder,” suggestive of preferential degradation of spacer DNA. Despite extensive loss of DNA and nuclear disintegration, the cell membrane of CAM-treated S-phase cells remains intact for several hours, excluding trypan blue or propidium iodide. Mitochondria, assayed for their ability to maintain a transmembrane potential (rhodamine 123 retention), as well as the lysosomal proton pump (probed by supravital uptake of acridine orange) also remain unchanged in these cells. G1 cells are refractory to CAM under these conditions. Synchronization of cells in S phase by aphidicolin increases the sensitivity of the whole cell population to CAM. The data suggest that CAM or other topoisomerase I inhibitors may be effective in some myelogenous leukemias, especially in combination with treatments synchronizing cells in S phase.

Phosphorylation of Replication Protein A: A Role for cdc2 Kinase in G1/S Regulation

Among the various phases of the cell cycle, we have concentrated on the regulation of the S phase. Our approach is to define biochemically the components of the DNA replication apparatus and then to analyze how the various components are regulated as cells enter, execute, and exit the S phase. Working from the substrate, namely the DNA replication apparatus, backward to the regulatory molecules would complement the more common genetic approach, which first uncovers the regulatory molecules and then works forward to the regulated substrates. Fusion of animal cells at various stages in the cell cycle has shown that S-phase nuclei contain a positive activator of DNA replication which is absent in G1 cells and which could hasten the onset of S phase in G1 nuclei (Rao and Johnson 1970). Hence, one of our goals is to identify biochemically this positive activator, tentatively designated S-phase promoting factor (SPF). Furthermore, once...

Caffeine overcomes a restriction point associated with DNA replication, but does not accelerate mitosis.

Mitotic chromosome condensation is normally dependent on the previous completion of replication. Caffeine spectacularly deranges cell cycle controls after DNA polymerase inhibition or DNA damage; it induces the condensation, in cells that have not completed replication, of fragmented nuclear structures, analogous to the S-phase prematurely condensed chromosomes seen when replicating cells are fused with mitotic cells. Caffeine has been reported to induce S-phase condensation in cells where replication is arrested, by accelerating cell cycle progression as well as by uncoupling it from replication; for, in BHK or CHO hamster cells arrested in early S-phase and given caffeine, condensed chromosomes appear well before the normal time at which mitosis occurs in cells released from arrest. However, we have found that this apparent acceleration depends on the technique of synchrony and cell line employed. In other cells, and in synchronized hamster cells where the cycle has not been subjected to prolonged continual arrest, condensation in replication-arrested cells given caffeine occurs at the same time as normal mitosis in parallel populations where replication is allowed to proceed. This caffeine-induced condensation is therefore "premature" with respect to the chromatin structure of the S-phase nucleus, but not with respect to the timing of the normal cycle. Caffeine in replication-arrested cells thus overcomes the restriction on the formation of mitotic condensing factors that is normally imposed during DNA replication, but does not accelerate the timing of condensation unless cycle controls have previously been disturbed by synchronization procedures.

Immunocytochemical determination of ploidy class-dependent bromodeoxyuridine incorporation in rat liver parenchymal cells after partial hepatectomy

Immunocytochemistry of bromodeoxyuridine (BrdU) incorporated in DNA was performed on cryostat sections of rat liver and on isolated hepatocytes after partial hepatectomy using a two-step labeling technique. The method enabled the detection of S-phase nuclei in both tissue preparations. Quantification of the number of labeled nuclei in sections showed that the number of nuclei in S-phase increased from 0.3% in control liver to about 36% at 24 h after partial hepatectomy. The detection of BrdU in isolated hepatocytes showed the same labeling index of binuclear diploid, mononuclear tetraploid and binuclear tetraploid cells. A special role for mononuclear diploid cells in proliferation did not seem to occur.

Changes in the nuclear distribution of DNA polymerase alpha and PCNA/cyclin during the progress of the cell cycle, in a cell-free extract of Xenopus eggs

The nuclear distribution of DNA polymerase alpha and PCNA/cyclin in embryonic nuclei has been investigated, in a cell-free extract of Xenopus eggs that recapitulates a basic cell-cycle in vitro, by indirect immunofluorescence microscopy. Both antigens co-distribute with the chromatin in S-phase nuclei; however, as DNA replication is completed and nuclei progress into a G2 state anti-PCNA fluorescence disappears and anti-DNA polymerase alpha fluorescence becomes resolved into bright spots. These spots are initially associated with the chromatin strands and can be seen to share both anti-PCNA and anti-DNA polymerase alpha fluorescence, but as anti-PCNA fluorescence fades the spots become dissociated from the chromatin and are redistributed throughout the nucleus until they are dispersed during nuclear envelope breakdown. The loss of anti-PCNA fluorescence and displacement of anti-DNA polymerase alpha fluorescence from the chromatin can be prevented by inhibiting DNA synthesis with aphidicolin. Under these conditions both antigens remain associated with the chromatin even after nuclear envelope breakdown and lamin dispersal. The association of these antigens with mitotic figures appears to be functional, as both biotin-11-dUTP and [32P]dCTP can be incorporated efficiently into DNA during the mitotic period.

Rapid Detection of Proliferating Cells Using Microwave Fixation and a Monoclonal Antibody to Bromodeoxyuridine

The objective of this research was to develop a rapid morphological test for proliferating cells using diaminobenzidine as the substrate marker. This technique can be employed to visualize proliferating cells by using a monoclonal antibody to bromodeoxyuridine and a standard immunoperoxidase technique. This paper describes methods for the rapid demonstration of S-phase nuclei in routine paraffin tissue sections. The tissue was fixed by exposure to microwave irradiation for various times and temperatures. The subsequent staining results are reliable and can easily fit into an existing immunoperoxidase program. (The J Histotechnol 12:101, 1989.)

Development of a tracheal implant xenograft model to expose human bronchial epithelial cells to toxic gases.

A tracheal implant model was developed which enabled exposure of differentiated normal human bronchial epithelial cells (NHBE) to a single exposure of a model toxic gas (formaldehyde). NHBE cells were grown in vitro in explant culture under defined serum-free conditions from previously frozen bronchial segments, harvested, and used to repopulate de-epithelialized rabbit tracheas. Rabbit tracheal segments repopulated with NHBE cells were implanted into the subcutis of congenitally athymic nude mice. Following graft vascularization, the xenografted NHBE cells differentiated and formed a mucociliary epithelial surface which lined approximately 50% of the surface of the implant lumen. Eight weeks post-implantation both ends of the implanted grafts were cannulated, and formaldehyde (HCHO) vapor (0, 6, or 15 ppm) in humidified air was passed through the tracheal lumens. Representative epithelial surfaces were examined by light and scanning electronmicroscopy, and autoradiography prior to, immediately after, and 48 hr following a 1-hr exposure to the test vapors. Light microscopic examination of implant sections immediately following exposure to 6 and 15 ppm HCHO detected cessation of ciliary activity, which recovered by 48 hr post-exposure. Scanning electron microscopic examination of the epithelial surface demonstrated mild morphologic changes, restricted to those implants exposed to 15 ppm. Findings immediately following HCHO exposure included swelling and exfoliation of individual cells, deciliation, and mucus release. Changes present after 48 hr included presence of flattened cells with few short microvilli and focal increase in the number of S-phase nuclei in the basal epithelium. These results demonstrate the utility of tracheal implants for single short-term exposure of differentiated human bronchial epithelial cells to gaseous agents.(ABSTRACT TRUNCATED AT 250 WORDS)

The control of DNA replication in a cell-free extract that recapitulates a basic cell cycle in vitro.

Cell-free extracts prepared from Xenopus eggs support chromosome decondensation and pronuclear formation on demembranated sperm heads. 32P-dCTP pulse-labelling studies demonstrate that DNA synthesis occurs in multiple bursts of 30-40 min in extracts containing pronuclei, each burst being followed by a period of 20-50 min during which no synthesis occurs. Density substitution with bromodeoxyuridine indicates that the synthesis in each burst is semiconservative and results from new initiations, and that, following multiple bursts of synthesis, reinitiation events can occur. Changes in nuclear morphology have been characterized in the extract by phase-contrast microscopy and by fluorescence microscopy following pulse labelling with biotin-11-dUTP and staining with anti-lamin antibodies. Lamin accumulation occurs as DNA decondenses and parallels the acquisition of membrane structures. Biotin-11-dUTP incorporation is first observed in small nuclei having decondensed DNA and an extensive lamina. While DNA synthesis is occurring nuclei remain relatively small, but rapid swelling accompanied by chromosome condensation occurs when biotin incorporation ceases. Nuclear swelling and chromatin condensation is followed by nuclear membrane breakdown, lamin dispersal and chromosome formation. Mitosis lasts for approximately 20 min. Nuclear reassembly is recognized by the appearance of membrane vesicles around small pieces of decondensed DNA, which parallels the appearance of lamin islands within a chromatin mass. These 'islands' incorporate biotin, indicating that DNA synthesis is occurring, and apparently fuse as larger S-phase nuclei are formed. Extensive protein synthesis occurs for at least 4 h in most extracts. This synthesis is required for the initiation of mitotic events and the reinitiation of DNA synthesis.

Changing spatial patterns of DNA replication in the developing wing of Drosophila

Using an antibody against bromodeoxyuridine we have analyzed the distribution of S-phase nuclei in the wing disc of Drosophila as the larval disc transforms into the adult wing during metamorphosis. On the basis of the timing of replication three cell populations can be distinguished: the cells of the presumptive wing margin, the precursor cells of the longitudinal veins, and those of the intervein regions. In each of these populations the cell cycle is first arrested and later resumes at a specific time, so that at each developmental time point a characteristic spatial pattern of S-phase nuclei is seen. An interpretation of these changing patterns in terms of vein formation, compartments, and neural development is offered.

A cell-cycle-dependent DNA polymerase activity that replicates intact DNA in chromatin

An insoluble DNA polymerase activity that replicates the intact chromatin template at 85% of the rate found in vivo has been partially characterized. HeLa cells, encapsulated in agarose microbeads, are lysed using an isotonic salt concentration: the resulting encapsulated nuclei contain polymerase associated with a nucleoskeleton and the unbroken template. This preparation can be manipulated freely without aggregation or breaking the DNA and yet is accessible to enzymes and other probes. The major activity, which is sensitive to aphidicolin, is found only in S-phase nuclei and replicates DNA semiconservatively, forming intermediates that are ligated efficiently into larger products.

Different populations of DNA polymerase α in HeLa cells

Three different populations of HeLa DNA polymerase α have been distinguished using a novel preparation of chromatin isolated using an isotonic salt concentration, which contains intact DNA. One synthesizes DNA in vitro at 85% of the rate in vivo, is found only in S-phase nuclei tightly associated with the nucleoskeleton and requires unbroken DNA in the form of chromatin as a template: we assume this is the authentic S-phase activity. On incubation at 37 °C, this activity dissociates from the nucleoskeleton to give a soluble activity that prefers broken templates. This soluble activity is in turn heterogeneous, containing active complexes of about 0.75 × 10 6 and 3 × 10 6 M r. The third activity is also soluble and released by lysing cells at any stage of the cell cycle. It, too, prefers broken templates. The authentic activity is obscured by the soluble ones if broken templates are provided.

Unscheduled DNA synthesis in lens epithelium after in vivo exposure to UV radiation in the 300 nm wavelength region.

A quantitative autoradiographic method was developed to study the pattern of DNA synthesis in the rat lens epithelium after in vivo exposure to UV radiation in the 300 nm wavelength region. It was found that UV radiation (peak transmission = 298 nm, half-width = +/- 10 nm) induces unscheduled DNA synthesis and that the proportion of nuclei in S-phase concurrently is reduced indicating an inhibition of the scheduled DNA synthesis. The registered unscheduled DNA synthesis is believed to be excision repair of DNA damage induced by the UV radiation. Excision repair in lens epithelial cells could be one mechanism involved in the correlation between exposure to sunlight and cataracts.

DNA replication and H5 histone exchange during reactivation of chick erythrocyte nuclei in heterokaryons.

Fusion of terminally differentiated chick erythrocytes (CE) with replicating quail myoblasts or established L6J1 rat myoblasts results in reactivation of DNA synthesis in the dormant CE nuclei and in suppression of DNA synthesis in the myoblast nuclei. The nuclei of primary quail myoblasts are more effectively inhibited than the nuclei of established rat myoblasts. Inhibition of DNA replication occurs not only by preventing G1 nuclei from entering S-phase but also by blocking nuclei in S-phase and by delaying nuclei in G2 from undergoing mitosis and starting a new DNA replication cycle. No inhibition of DNA synthesis could be observed when mouse erythrocytes, i.e., erythrocytes lacking nuclei, were fused with rat myoblasts to generate mouse-globin-containing L6J1 cybrids.--Reactivation of CE nuclei is associated with a loss of the tissue-specific H5 histone variant. Complete elimination of H5 histone, however, does not seem to be a necessary prerequisite for the initiation or completion of DNA replication in CE nuclei since H5 antigens are found on reactivated G1, S, and G2 nuclei.

Cis-diamminedichloroplatinum-mediated crosslinking of nuclear proteins to DNA is cell cycle specific

Immunochemical analysis was employed to investigate the cell cycle-dependent protein-DNA crosslinking by cis-diamminedichloroplatinum II ( cis-DDP), in HeLa-S 3 cells. Cells synchronized by double thymidine block or hydroxyurea were released into S phase and incubated at 2-h intervals with cis-DDP as they progressed through S 1, G 2, M, and then into G 1 and S phases of the subsequent cycle. Immunoblots of the DNA-crosslinked antigens reacted with antisera to 0.35 m NaCl extract or residue of HeLa S-phase nuclei revealed that several antigens changed their DNA-crosslinking pattern during the progression of HeLa cells through their reproductive cycle.

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 G 1 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 G 1 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 G 1- and S-phase cells, respectively. The G 1 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 G 1 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.

DNA repair synthesis in cultured mammalian and fish cells following exposure to chemical mutagens

Unscheduled DNA repair synthesis (UDS) was measured autoradiographically in HF, CHO, RTG, RTO, CH and FHM cells given a 3-h exposure to MNNG, 4NQO, NA2AAF and AFB 1. All the chemicals produced a dose-response, the magnitude of which varied with the particular chemical and cell line. HF produced the greatest response, CHO less and the fish cell lines the least. The response of all fish cell lines was approximately equal for a particular chemical. A number of factors were investigated to account for the comparative differences in UDS response. The time course of repair in HF, CHO and RTG following a 3-h exposure to MNNG or 4NQO was the same. As S-phase nuclei were observed in control slides and the amount of repair following UV exposure varies HF > CHO > RTG, neither 3HTdR nor mutagen uptake is limiting. The observed results are discussed.

Control of histone gene expression in Physarum polycephalum. I. Protein synthesis during the cell cycle.

Synchronous cultures of Physarum polycephalum were pulsed with [3H]lysine hydrochloride in S and G2 phases of the cell cycle. Plasmodial extracts were separated into nuclear, ribosomal and acid-soluble post-ribosomal cytoplasmic fractions. Core histones could be detected by staining in the nuclear fractions of both S and G2 phases, but were not detected by staining in the cytoplasmic fractions. Newly synthesized histone was present in S-phase nuclei but not in S-phase cytoplasm. The specific activity of newly synthesized histone in G2-phase nuclei decreased by at least 95% compared to S phase and no newly synthesized histone was observed in G2-phase cytoplasmic fractions. Thus histone synthesis is restricted to S phase. There are no free pools of histone in the cytoplasm of Physarum in either S or G2 phases of the cell cycle.