Lamin disassembly kinetics: A cell-free system with extracts from mitotic HeLa cells

We describe a cell-free system in which a postribosomal supernatant from metaphase HeLa cells induces prophase-like changes in permeabilized HeLa cell populations as evidenced by the nuclear lamin disassembly and chromatin condensation. We have attempted to characterize the cell-free system with permeabilized HeLa cells. First, by extracting lamins with agents known to disrupt the noncovalent interactions in the supramolecular lamin aggregate in interphase using polyclonal and a newly established monoclonal anti-lamin Ab 2E3, uniform extraction of lamins was achieved with urea and deoxycholate whereas the cation Mg2+ and 2-mercaptoethanol had little effect on the disassembly of interphase lamins. Second, cytoplasmic extract from mitotic HeLa cells, synchronized by a nitrous oxide metaphase arrest, was tested. It had a differential effect on interphase lamin depolymerization. Nuclei in G1 phase of the cell cycle were more resistant against the mitotic extracts than cells in S and G2 phase. The results are discussed in terms of a possible inactivation of mitotic extracts by factors present in nuclei in early interphase.     

RIBONUCLEIC ACID AND PROTEIN SYNTHESIS IN MITOTIC HELA CELLS

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

Isolation of chromosome clusters from metaphase-arrested HeLa cells

We have developed a simplified approach for the isolation of metaphase chromosomes from HeLa cells. In this method, all the chromosomes from a cell remain together in a bundle which we call a metaphase chromosome cluster. Cells are arrested to 90–95% in metaphase, collected by centrifugation, extracted with non-ionic detergent in a low ionic strength buffer at neutral pH, and homogenised to strip away the cytoskeleton. The chromosome clusters which are released can then be isolated in a crude state by pelleting or they can be purified away from nearly all the interphase nuclei and cytoplasmic debris by banding in a Percoll^TM density gradient. — This procedure has the advantages that it is quick and easy, metaphase chromatin is recovered in high yield, and Ca⁺⁺ is not needed to stabilise the chromosomes. Although the method does not yield individual chromosomes, it is nevertheless very useful for both structural and biochemical studies of mitotic chromatin. The chromosome clusters also make possible biochemical and structural studies of what holds the different chromosomes together. Such information could be useful in improving chromosome isolation procedures and for understanding suprachromosomal organisation of the nucleus.     

Factors influencing ADP-ribosylation differences between chromosomal proteins of interphase and metaphase HeLa cells

Fundamental differences were previously discovered in the ADP-ribosylation of proteins from metaphase chromosomes and interphase nuclei of HeLa cells. The number of modified nonhistone species was found to be dramatically reduced for metaphase chromosomes. An investigation has therefore been made of factors which could influence, and therefore be responsible for, this change in ADP-ribosylation during the cell cycle. Modified proteins were detected by autoradiography of sodium dodecyl sulfate-polyacrylamide gels containing mitotic and interphase samples from permeabilized cells that had been incubated with [32P]NAD. Whole cells showed a difference between interphase and metaphase similar to that for isolated nuclei and chromosomes. Chromosome expansion, disruption of chromosomes or nuclei, DNA nicking, and cellular growth activity significantly changed the incorporation of 32P label. Inhibitors of protein, RNA, and DNA synthesis did not, however, greatly affect ADP-ribosylation. The pattern of labeled species was not altered by the presence of nonradioactive NAD, though the extent of labeling declined. The results were not artifactually due to the procedure used to arrest cells in mitosis. Similar results were found with Novikoff rat hepatoma cells, demonstrating that the difference between metaphase and interphase is not confined to HeLa cells.     

Metaphase arrest and cell death induced by etoposide on HeLa cells

DNA damage, cell cycle and apoptosis form a network with important implications for cancer chemotherapy. Dysfunctions of the cycle checkpoints can allow cancer cells to acquire drug resistance. Etoposide is a well-known inducer of apoptosis, which is widely used in cell biology and in clinical practice. In this work we report that a pulse of 50 μM etoposide (incubation for only 3 h) on HeLa cells causes a sequence of events that leads to abnormal mitotic figures that could be followed either by cell death or, more commonly, by interphase restitution and endocycle. The endocycling polyploid cells enter immediately into mitosis and suffer metaphase blockage with multiple spindle poles, which were generally followed by a direct triggering of apoptosis from metaphase (mitotic catastrophe), or by a new process of endocycling, until surviving cells finally became apoptotic (96 h after the treatment).     

Virus Replication, Cytopathology, and Lysosomal Enzyme Response of Mitotic and Interphase Hep-2 Cells Infected with Poliovirus

Mitotic Hep-2 cells, selected by the PEL (colloidal silica) density gradient method and held in mitosis with Colcemid, are readily infected by poliovirus type I (Mahoney). They produce and release the same amount of virus as interphase, random-growing cells. In contrast to interphase cells, mitotic cells show no detectable virus-induced cytopathic effect at the light microscopy level and only slight alterations, consisting of small clusters of vacuoles, at the electron microscopy level. Mitotic cells contain the same total amount of lysosomal enzymes per cell as interphase cells, but they display no redistribution of lysosomal enzymes during the virus infection as interphase cells do. This supports the view that lysosomal enzyme redistribution is associated with the cytopathic effect in poliovirus infection but shows that virus synthesis and release is not dependent on either the cytopathic effect or lysosomal enzyme release. The possible reasons for the lack of cytopathic effect in mitotic cells are discussed.     

Fragmentation of re-formation of mitotic Golgi apparatus detected by a centrifugal method

The fragmentation/re-formation process of the Golgi apparatus during mitosis was studied by flotation centrifugation in a stepwise sucrose density gradient. The mitotic Golgi fraction was obtained from Chinese hamster ovary cells synchronized with thymidine and nocodazole. The Golgi apparatus detected by a marker enzyme, galactosyltransferase, was separated into two peaks by the flotation centrifugation. The amount of the Golgi recovered at the lower density peak was less in the mitotic cells than in the interphase cells. The separation profile of the mitotic Golgi returned to that of the interphase Golgi by further incubation of the mitotic cells. The re-formation of the fragmented Golgi was inhibited by nocodazole and vinblastine, but not by actinomycin D and cycloheximide.     

PROPHASING OF INTERPHASE NUCLEI AND INDUCTION OF NUCLEAR ENVELOPES AROUND METAPHASE CHROMOSOMES IN HELA AND CHINESE HAMSTER HOMO- AND HETEROKARYONS

Fusing human HeLa metaphase cells with HeLa interphase cells resulted within 30 min in either of two phenomena in the resultant binucleate cell: either prophasing of the interphase nucleus or formation of a normal-appearing nuclear envelope around the metaphase chromosomes. The frequency of either occurrence was strongly dependent on environmental pH. At pH's of 6.6–8.0, prophasing predominated; at pH 8.5 nuclear envelope formation predominated. Additionally, the frequencies of the two events in multinucleate cells depended on the metaphase/interphase ratio. When the ratio was 0.33 nuclear envelope formation predominated; when it was 2.0 prophasing predominated. In their general features, the results with fused HeLa cells resembled those reported earlier with fused Chinese hamster Don cells. However, the results provided an indication that between pH 6.6 and 8.0 the HeLa metaphase cells possessed a much greater capacity than the Don metaphase cells to induce prophasing. Fusion of Don metaphase cells with HeLa interphase cells or of Don interphase cells with HeLa metaphase cells at pH 8.0 resulted in nuclear envelope formation or prophasing in each kind of heterokaryon. As in the homokaryons, the frequencies of the two events in the heterokaryons depended on the metaphase/interphase ratio. The statistics of prophasing and nuclear envelope formation in the homo- and heterokaryon populations were consistent with the notion that disruption or formation of the nuclear envelope depends on the balance attained between disruptive and formative processes.     

Evidence that globular Golgi clusters in mitotic HeLa cells are clustered tubular endosomes.

By conventional electron microscopy we observed in mitotic HeLa cells the structures termed Golgi clusters by Lucocq et al. (J. Cell Biol. 104, 865-874 (1987)) and interpreted by them as clusters of vesicular remnants of the Golgi apparatus. Golgi clusters consist of tubular and vesicular profiles about 50 nm in diameter, sometimes associated with larger 250 nm vesicles. When cultures of HeLa cells were incubated for 60 min or 120 min with medium containing high specific activity horseradish peroxidase (HRP) at 10 mg/ml we found that the membrane-bound compartments in the Golgi clusters in mitotic cells contained heavy deposits of HRP reaction product. Neither interphase nor mitotic HeLa cells contain an endogenous peroxidase activity. We concluded that Golgi clusters are an endocytic compartment and confirmed this by showing that Golgi clusters could be labeled with two other endocytic tracers--bovine serum albumin conjugated to colloidal gold and transferrin conjugated to HRP. When cultures were incubated with HRP for only 15 min most of the Golgi clusters in the mitotic cells were either unlabeled or consisted of a mixture of HRP-labeled and unlabeled profiles. Since during mitosis endocytosis is inhibited this was the expected result. When interphase HeLa cells were incubated with Brefeldin A (BFA), the Golgi apparatus disassembled and immunofluorescence microscopy showed that 1,4 beta galactosyltransferase had relocated to the endoplasmic reticulum. When cells in the presence of BFA and lacking the Golgi apparatus were allowed to endocytose HRP and then entered mitosis, typical HRP-labeled Golgi clusters were seen in the mitotic cells. It is therefore highly unlikely that these structures contain membrane derived from the Golgi cisternae that are sensitive to BFA, including in HeLa cells those containing galactosyltransferase. Finally, we found that interphase HeLa cells incubated with okadaic acid contain structures that are morphologically indistinguishable from Golgi clusters but can be labeled by endocytic tracer. Taken together, this evidence indicates that most, if not all, of the membrane-bound compartments in Golgi clusters are tubular early endosomes.     

Disorganization of mitosis in HeLa cells by deuterium oxide

We assessed, by light and electron microscopy, the influence of deuterium oxide on the dynamics of mitosis and on the morphology of the mitotic apparatus in HeLa cells grown in vitro. A 2-h incubation of HeLa monolayers with low concentrations of D2O (1%-25%) in the medium increased the frequency of multipolar divisions up to 20 times the control level. Substitution of 10% and 25% D2O for H2O induced changes in the proportions of mitotic phases. These changes could be fully reversed to the control pattern after 1 h of recovery in non-deuterated medium. Fifty % D2O strongly inhibited, and 75% D2O blocked the cell cycle before prophase and at (pro-)metaphase. In cells treated with 50% D2O conspicuous morphological changes of the interphase chromatin as well as ultrastructural abnormalities of all mitotic phases were regularly observed. Overall, these results confirmed the antimitotic activity of deuterium oxide and revealed that it could also influence the cell cycle before mitosis. It is suggested that interference with diverse cellular constituents rather than a specific influence on microtubule turnover could be responsible for the disorganization of the cell cycle in HeLa cells by D2O.     

Differential dynamics of splicing factor SC35 during the cell cycle

Pre-mRNA splicing factors are enriched in nuclear domains termed interchromatin granule clusters or nuclear speckles. During mitosis, nuclear speckles are disassembled by metaphase and reassembled in telophase in structures termed mitotic interchromatin granules (MIGs). We analysed the dynamics of the splicing factor SC35 in interphase and mitotic cells. In HeLa cells expressing green fluorescent protein (GFP)-SC35, this was localized in speckles during interphase and dispersed in metaphase. In telophase, GFP-SC35 was highly enriched within telophase nuclei and also detected in MIGs. Fluorescence recovery after photobleaching (FRAP) experiments revealed that the mobility of GFP-SC35 was distinct in different mitotic compartments. Interestingly, the mobility of GFP-SC35 was 3-fold higher in the cytoplasm of metaphase cells compared with interphase speckles, the nucleoplasm or MIGs. Treatment of cells with inhibitors of cyclin-dependent kinases (cdks) caused changes in the organization of nuclear compartments such as nuclear speckles and nucleoli, with corresponding changes in the mobility of GFP-SC35 and GFP-fibrillarin. Our results suggest that the dynamics of SC35 are significantly influenced by the organization of the compartment in which it is localized during the cell cycle.     

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.     

Enucleation of cells by density gradient centrifugation

HEp-2 cells can be enucleated by ultracentrifugation in a colloidal silica (PTL) density gradient, containing cytochalasin B. Under optimal conditions, more than 70% of the cells are enucleated. Purification up to 97% is carried out by centrifugation at low speed through a second, preformed PTL density gradient. The enucleated cells show a high viability, as tested by [³H]leucine incorporation. The method described was developed for enucleation of high quantities of cells and has the advantage that it can be used for cell types which do not adhere firmly enough to a carrier to be centrifuged as a monolayer.     

Nuclear lamins during prophasing and telophasing in heterophasic HeLa and Chinese hamster homokaryons

We have perturbed the dynamics of the nuclear lamins by means of cell fusion between mitotic and interphase cells and have studied redistribution of lamins in fused cells as a function of extracellular pH levels. We show here that in heterophasic M-1 HeLa homokaryons disassembly of interphase lamins predominates at low pH levels between 7.0 to 7.3, whereas deposition of cytoplasmic lamins around condensed metaphase chromosomes was observed at pH 8.0. In HeLa homokaryons lamina disassembly and lamina deposition around chromosomes are mutually exclusive. Using heterophasic M-1 homokaryons of the Chinese hamster cell line DON we observed that disassembly of interphase lamins and deposition of lamins around condensed chromosomes coexisted in the same homokaryon kept at pH 7.0. Disassembly of lamins developed synchronously with premature chromosome condensation (PCC) whereas lamina deposition around the condensed M-chromosomes was followed by telophasing. In fusions kept at pH 8.0 cytoplasmic lamins were exclusively deposited around mitotic chromosomes. The results are interpreted as showing that pH regulates the lamina dynamics in homokaryons of mitotic and interphase cells.     

Specific attachment of nuclear-mitotic apparatus protein to metaphase chromosomes and mitotic spindle poles: possible function in nuclear reassembly

NuMA protein is the largest, abundant, primate-specific chromosomal protein. The protein was purified from HeLa cells and monospecific monoclonal antibodies were prepared that react exclusively with NuMA protein in immunoblot analysis. These antibodies were used to define the intracellular location and properties of NuMA protein. Using indirect immunofluorescence, NuMA protein was detected only in the nucleus of interphase cells and on the chromosomes in mitotic cells. One class of monoclonal antibody called the 2E4-type antibody, caused NuMA protein (or a complex of proteins including NuMA) to be released from its binding site on metaphase or anaphase chromosomes. The separation of NuMA protein from chromosomes was observed either with the immunofluorescence assay or in electrophoretic analyses of proteins released from isolated metaphase chromosomes after reaction with 2E4 antibody. The immunofluorescence studies also showed that after release of the NuMA protein from chromosomes of metaphase or anaphase cells, the protein bound specifically to the polar region of the mitotic spindle. It was shown that exogenously added NuMA antigen/antibody complex bound only to the mitotic spindle poles of permeabilized primate cells and not to the spindle poles of other mammalian cells, thus demonstrating the specificity of the spindle-pole interaction. The antibody mediated transfer of NuMA from chromosomes to poles was blocked when the chromosomes were treated with cross-linking fixatives. Results suggest that the NuMA protein has specific attachment sites on both metaphase chromosomes and mitotic spindle poles (the site where post-mitotic nuclear assembly occurs). A model is proposed suggesting that a protein having such dual binding sites could function during nuclear reassembly to link mitotic chromosomes into the reforming nucleus.     

Time-Lapse Cinemicrographic Studies of X-Irradiated HeLa S3 Cells: I. Cell Progression and Cell Disintegration

Time-lapse cinemicrographs of synchronous HeLa S3 cells irradiated with 220 kv X-rays at various stages of interphase provided data for constructing pedigrees, measuring the duration of both generation cycles and mitoses, and scoring events associated with cell disintegration for up to seven postirradiation generations. The onset of the first mitosis after doses of 500 rads was delayed as expected from previous studies of the age dependence of “mitotic delay.” The interval between this first mitosis and the next was indistinguishable from that for unirradiated control cells, while the subsequent two generations were again prolonged, on the average, though not so severely as was the irradiated generation. The duration of mitosis was increased proportionally more than interphase. Cell disintegration took place by way of two morphologically distinct processes. In three-quarters of the cases the cells were rounded and apparently trapped in metaphase when they disintegrated; the remaining disintegrations occurred in spread, interphase cells. In cells disintegrating from the rounded configuration, the generation preceding disintegration was prolonged relative to that in cells which divided; in cells disintegrating from either configuration, the penultimate generation was also prolonged. The mitotic times were disproportionately increased in both of these generations. It is suggested that in this system X-ray damage is preferentially expressed as derangement of the mitotic process; such damage ultimately brings about permanent mitotic arrest in the majority of cells.     

Different sensitivity of DNA in situ in interphase and metaphase chromatin to heat denaturation

Heat denaturation of DNA in situ, in unbroken cells, was studied in relation to the cell cycle. DNA in metaphase cells denatured at lower temperatures (8 degrees-10 degrees C lower) than DNA in interphase cells. Among interphase cells, small differences between G1, S, and G2 cells were observed at temperatures above 90 degrees C. The difference between metaphase and interphase cells increased after short pretreatment with formaldehyde, decreased when cells were heated in the presence of 1 mM MgCl2, and was abolished by cell pretreatment with 0.5 N HCl. The results suggest that acid-soluble constituents of chromatin confer local stability to DNA and that the degree of stabilization is lower in metaphase chromosomes than in interphase nuclei. These in situ results remain in contrast to the published data showing no difference in DNA denaturation in chromatin isolated from interphase and metaphase cells. It is likely that factors exist which influence the stability of DNA in situ are associated with the super-structural organization of chromatin in intact nuclei and which are lost during chromatin isolation and solubilization. Since DNA denaturation is assayed after cell cooling, there is also a possibility that the extent of denatured DNA may be influenced by some factors that control strand separation and DNA reassociation. The different stainability of interphase vs. metaphase cells, based on the difference in stability of DNA, offers a method for determining mitotic indices by flow cytofluorometry, and a possible new parameter for sorting cells in metaphase.     

Fractionation of cells and subcellular particles with Percoll

At present, centrifugation is the most common method for separation and isolation of cells and subcellular particles. The technique can be used for a wide range of applications. During latter years it has become obvious what a powerful method density gradient centrifugation is, especially when used in conjunction with sensitive assays or clinical treatments. The most active areas for use of density gradient centrifugation include purification for in vitro fertilization of sperm of both human and bovine origin, isolation of cells for cell therapy of patients receiving chemo- and radiation therapy and basic research both on cellular and subcellular levels. These treatments and investigations require homogeneous populations of cells and cell organelles, which are undamaged after the separation procedure. Percoll, once introduced to reduce convection during centrifugation, has proved to be the density gradient medium of choice since it fulfills almost all criteria of an ideal density gradient medium. Recently good results have also been obtained after silanization of colloidal silica particles, e.g. BactXtractor. The latter medium has proved to be useful in recovery of microorganisms from food samples free of inhibitors to the Polymer Chain Reaction (PCR). The separation procedures described for Percoll in this review seem to be applicable to any cells or organelles in suspension for which differences in size or bouyant density exist. Furthermore, since Percoll media are inert, they are well suited for the separation of fragile elements like enveloped viruses.     

The viability of cells grown or centrifuged in a new density gradient medium, Percoll(TM)

A new density gradient medium, Percoll (a modified colloidal silica), has been tested for toxicity in primary cultures of rat liver and calf testicle cells, and in continuous cultures of pig kidney and HeLa cells. The presence of Percoll did not appreciably affect the growth or viability of the cells as judged from cell counts and morphology. The various cells were also centrifugea in gradients of Percoll and subsequently cultured. The in vitro growth of the cells was similar to that of untreated cells. Rat liver cells were labelled in vivo with [¹²⁵I]asialoceruloplasmin (parenchymal cells) or heat-denatured [¹²⁵I]albumin (non-parenchymal cells). After dispersion of the cells and iso-pycnic centrifugation in Percoll the non-parenchymal cells banded preferentially at a lower density (1.04−1.05 g/ml) than parenchymal cells (1.07−1.09 g/ml). The two types of cells showed very different morphology in cell culture. The non-parenchymal cells retained their phagocytic properties during culture. Injured cells and cell debris band at the top of the Percoll gradients in contrast to their behaviour in gradients containing low molecular weight substances. Centrifugation in Percoll can be used to enrich viable cells.     

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

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