Molecular mechanisms of the chromosome condensation and decondensation cycle in mammalian cells

The chromosomes undergo a condensation-decondensation cycle within the life cycle of mammalian cells. Chromosome condensation is a complex and critical event that is necessary for the equal distribution of genetic material between the two daughter cells. Although chromosome condensation-decondensation and segregation is mechanistically complex, it proceeds with high fidelity during the eukaryotic cell division cycle. Cell fusion studies have indicated the presence of chromosome condensation factors in mammalian cells during mitosis. If extracts from mitotic cells are injected into immature oocytes of Xenopus laevis, they induce meiotic maturation (i.e. germinal vesicle breakdown and chromosome condensation) within 2–3 hours. Recently, we showed that the maturation-promoting activity of the mitotic cell extracts is inactivated by certain protein factors present in cells during the G₁ period. The activity of the G₁ factors coincides with the process of chromosome decondensation that begins at telophase and continues throughout the G₁ period. These studies have revealed that the mitotic factors and the G₁ factors play a pivotal role in the regulation of condensation and decondensation of chromosomes. Furthermore, our studies strongly suggest that nonhistone protein phosphorylation and dephosphorylation may mediate chromosome condensation and decondensation, respectively.     

Mitotic chromosome size scaling in Xenopus

As rapid divisions without growth generate progressively smaller cells within an embryo, mitotic chromosomes must also decrease in size to permit their proper segregation, but this scaling phenomenon is poorly understood. We demonstrated previously that nuclear and spindle size scale between egg extracts of the related frog species Xenopus tropicalis and Xenopus laevis but show here that dimensions of isolated mitotic sperm chromosomes do not differ. This is consistent with the hypothesis that chromosome scaling does not occur in early embryonic development when cell and spindle sizes are large and anaphase B segregates chromosomes long distances. To recapitulate chromosome scaling during development, we combined nuclei isolated from different stage Xenopus laevis embryos with metaphase-arrested egg extracts. Mitotic chromosomes derived from nuclei of cleaving embryos through the blastula stage were similar in size to replicated sperm chromosomes but decreased in area approximately 50% by the neurula stage, reproducing the trend in size changes observed in fixed embryos. Allowing G₂ nuclei to swell in interphase prior to mitotic condensation did not increase mitotic chromosome size, but progression through a full cell cycle in egg extract did, suggesting that epigenetic mechanisms determining chromosome size can be altered during DNA replication. Comparison of different sized mitotic chromosomes assembled in vitro provides a tractable system to elucidate underlying molecular mechanisms.Key words: mitotic chromosomes, Xenopus, egg extracts, intracellular scaling, spindle, embryogenesis, cell division     

CDK1 Inactivation Regulates Anaphase Spindle Dynamics and Cytokinesis In Vivo

Through association with CDK1, cyclin B accumulation and destruction govern the G₂/M/G₁ transitions in eukaryotic cells. To identify CDK1 inactivation-dependent events during late mitosis, we expressed a nondestructible form of cyclin B (cyclin BΔ90) by microinjecting its mRNA into prometaphase normal rat kidney cells. The injection inhibited chromosome decondensation and nuclear envelope formation. Chromosome disjunction occurred normally, but anaphase-like movement persisted until the chromosomes reached the cell periphery, whereupon they often somersaulted and returned to the cell center. Injection of rhodamine-tubulin showed that this movement occurred in the absence of a central anaphase spindle. In 82% of cells cytokinesis was inhibited; the remainder split themselves into two parts in a process reminiscent of Dictyostelium cytofission. In all cells injected, F-actin and myosin II were diffusely localized with no detectable organization at the equator. Our results suggest that a primary effect of CDK1 inactivation is on spindle dynamics that regulate chromosome movement and cytokinesis. Prolonged CDK1 activity may prevent cytokinesis through inhibiting midzone microtubule formation, the behavior of proteins such as TD60, or through the phosphorylation of myosin II regulatory light chain.     

Nuclear phosphoinositide 3-kinase C2β activation during G2/M phase of the cell cycle in HL-60 cells

The activity of nuclear phosphoinositide 3-kinase C2β (PI3K-C2β) was investigated in HL-60 cells blocked by aphidicolin at G₁/S boundary and allowed to progress synchronously through the cell cycle. The activity of immunoprecipitated PI3K-C2β in the nuclei and nuclear envelopes showed peak activity at 8 h after release from the G₁/S block, which correlates with G₂/M phase of the cell cycle. In the nuclei and nuclear envelopes isolated from HL-60 cells at 8 h after release from G₁/S block, a significant increase in the level of incorporation of radiolabeled phosphate into phosphatidylinositol 3-phosphate (PtdIns(3)P) was observed with no change in the level of radiolabeled PtdIns(4)P, PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃. On Western blots, PI3K-C2β revealed a single immunoreactive band of 180 kDa, whereas in the nuclei and nuclear envelopes isolated at 8 h after release, the gel shift of 18 kDa was observed. When nuclear envelopes were treated for 20 min with μ-calpain in vitro, the similar gel shift and increase in PI3K-C2β activity was observed which was completely inhibited by pretreatment with calpain inhibitor calpeptin. The presence of PI3K inhibitor LY 294002 completely abolished the calpain-mediated increase in the activity of PI3K-C2β but did not prevent the gel shift. When HL-60 cells were released from G₁/S block in the presence of either calpeptin or LY 294002, the activation of nuclear PI3K-C2β was completely inhibited. These results demonstrate the calpain-mediated activation of the nuclear PI3K-C2β during G₂/M phase of the cell cycle in HL-60 cells.     

Cell cycle analysis in a multinucleate green alga,Boergesenia forbesii (Siphonocladales,Chlorophyta)*

The cell cycle (nuclear division cycle) of a multinucleate green alga, Boergesenia forbesii (Harvey) Feldmann was studied using microspectrophotometry and BrdU incorporation techniques. Mitosis was observed frequently 1-4 h after the beginning of the light period, on a 16:8 h LD cycle at 25°C. Mitotic nuclei formed discrete patches. Other nuclei remained in the G₁ period. The DNA synthetic phase (S phase) was estimated to last about 12 h from microspectrophotometric study using aphidicolin inhibition just before the S phase and release from it. The G₂ period was estimated to be about 2 h, because a labeled prophase nucleus could be detected when the samples were labeled with BrdU continuously over 3 h. The incorporation pattern of BrdU changed through the S phase nucleus. In early S phase, BrdU staining was detected as many dots in the entire nucleus, while in late S phase, it was detected as several discrete regions along the nuclear membrane. Almost all nuclei in B. forbesii were in the G₁ stage after nuclear division, and the nuclei in several patches of the cell simultaneously initiated DNA synthesis. Once the nuclei entered into S phase, these nuclei continued into G₂ and mitosis. In other words, the cell cycle regulation of entrance into S phase from G₁ is an important factor in the growth and morphogenesis in B. forbesii.     

Change of chromatin morphology during the cell cycle detected by means of automated image analysis

Mouse embryo fibroblasts growing asynchronously in vitro stained with Feulgen method and their nuclear chromatin was analysed by means of the image analysing computer Quantimet 720D. Cells with 2C, 3C and 4C content of DNA were considered as being in G₁, middle S and G₂ phase of cell cycle, respectively. It was found that the projected area of nuclei increases during the cell cycle and that the mean optical density of chromatin increases from G₁ through S to G₂ phase. The curves showing the areas of chromatin at different optical density thresholds are different for cells in G₁, S and G₂ phase. The results demonstrate cyclic changes in chromatin morphology in the interphase nuclei during the cell cycle.     

CELLULAR AND NUCLEAR VOLUME DURING THE CELL CYCLE OF NHIK 3025 CELLS

The distribution of cellular and nuclear volume in synchronous populations of NHIK 3025 cells, which derive from a cervix carcinoma, have been measured by electronic sizing during the first cell cycle after mitotic selection. Cells given an X-ray dose of 580 rad in G₁, were also studied. During the entire cell cycle the volume distribution of both cells and nuclei is an approximately Gaussian peak with a relative width at half maximum of about 30%. About half of this width is due to imperfect synchrony whereas the rest is associated with various time invariant factors. During S the mean volume of the cells grows exponentially whereas the nuclear volume increases faster than for exponential kinetics. Hence, although cellular and nuclear volumes are closely correlated, their ratio does not remain constant during the cell cycle. Volume growth during the first half of G₁ is negligible especially for nuclei where the growth appears to be closely associated with DNA-synthesis. For unirradiated cells the growth of cellular and nuclear volume is negligible also during G₂+ M. In contrast, the X-irradiated cells continue to grow during the 6 hr mitotic delay with a rate that is constant and about half of that observed in late S. Hence, radiation induced mitotic delay does not appear merely as a lengthening of an otherwise normal G₂. During G₁ and S the irradiated cells were identical to unirradiated ones with respect to all the parameters measured.     

The chromosomes of Puschkinia libanotica during mitosis

The chromosome complement of Puschkinia libanotica is described. In addition to five pairs of A chromosomes plants may possess up to 7 B chromosomes. Part of the long arm of the B chromosome gives rise to a heterochromatic mass in interphase nuclei and this can be seen to be a double structure in G₁ nuclei and a quadruple structure in G₂ nuclei. It is believed that these configurations represent the pre- and post-replication forms of subchromatids in the heterochromatic segment of the B chromosome. Microdensitometry of metaphase chromosomes shows that the segment of the B chromosome that is heterochromatic during interphase has no more DNA per unit volume than any of the euchromatic A chromosomes.     

Promotive and inhibitory effects of diverse arabinogalactan proteins on Daucus carota L. somatic embryogenesis

In the 3-d-old 2-mm root tip of Pisum sativum L. cv. Lincoln the percentage of actively proliferating cells is estimated to be 70%. The remaining cells are non-cycling and arrested with 2C and 4C DNA content in G₀ and in G₂Q, respectively. In this work we studied the kinetic significance of these quiescent cells, using the sorting capabilities of flow cytometry and immunofluorescence techniques to detect the proliferation marker PCNA (proliferating cell nuclear antigen) inside cells within the different cell-cycle compartments. While in animal cells, PCNA is present at a high level only in actively proliferating cells, in 3-d-old pea root tips 95% of the cells are PCNA-positive. After flow cytometry and sorting of pea non-cycling nuclear populations, all G₂Q nuclei appeared strongly PCNA-positive, indicating that these cells had recently left the cell cycle. By contrast, most G₀ nuclei showed a low level of PCNA immunofluorescence intensity, as measured by image analysis, with about 25% of the nuclei being PCNA-negative. This small percentage was found to correspond to root cap cells, as could be observed in the root tip section. These are the only cells in the root apical region which are fully differentiated and which, therefore, lack the competence to enter the cell cycle. In contrast, the more or less PCNA-positive G₀ nuclei could represent a kinetically heterogeneous population of cells competent to proliferate, but which have either recently left the cell cycle or are progressing to the G₀-G₁ transition. Received: 6 November 1996 / Accepted: 14 January 1997     

Nuclear antigens in the HeLa cell cycle

Summary Antisera to 0.35 M NaCl extracts and residues of S phase HeLa nuclei were reacted with electrophoretically separated proteins from the nuclei or nuclear material of HeLa cells synchronized in G₁, S, G₂ or M phases of the cell cycle. Quantitative evaluation of the peroxidase-antiperoxidase stained nitrocellulose transfers (Western blots) revealed significant changes in the quantities of nuclear non-histone proteins during the cell cycle. Immunochemical staining of electrophoretically separated nuclear antigens permits their selective detection in minute quantities and in the presence of many additional proteins.     

Altered cell cycle progression and aberrant mitosis in adenovirus-infected rodent cells

Actively growing mouse or rat embryo cells suffered structural chromosome damage, mitotic anomalies, and polyploidy after infection by human adenovirus type 5. Chromosome damage required expression of one or more early viral genes and showed regular periodicity in its frequency. The growth cycle time of some of the infected cells was reduced by about 5 hours due to a decrease in G₁, and the interval between successive waves of chromosome damage corresponded to this reduced cycle time. After infection there was a decrease in cells with G₁ DNA content and an increase in cells with G₂ diploid, aneuploid, and polyploid DNA contents. We suggest these effects are due to the expression in semipermissive cells of early viral gene(s), whose function in productive infection in vivo is to alter cell cycle controls in order to maximize the number of cells able to replicate viral DNA and the time such cells spend in DNA replication.     

Chronology and pattern of replication in the bone marrow chromosomes of Gallus domesticus

The duration of the cell generation, the chronology, and the pattern of chromosome duplication was studied in the bone marrow of Gallus domesticus. The duration of the phases of the cell cycle is: cell generation 17.5 hours, S period 9 hours. G₂ period plus prophase stage 2.5 hours, G₁ period 6 hours. Chromosome replication begins at many sites. During middle S it extends to the whole complement and finally finishes in small, late replicating regions of the macrochromosomes. Interchromosomal asynchrony of duplication at the initiation or at the end of the S period was not observed. Z-chromosomes begin and finish DNA synthesis synchronously with the other macrochromosomes. The W-chromosome in females is the last microchromosome to finish replication. However it ends DNA synthesis at about the same time as the macrochromosomes. Similarities and differences between chromosome replication in Aves and Mammalia are considered.     

Preservation of chromosome integrity during micronucleation induced by colchicine in PtK1 cells

Colchicine induces the formation of small nuclei called micronuclei which contain limited parts of the genome. Some of them exhibit a DNA content equivalent to that of a single chromosome. Our purpose was to study the preservation of chromosome integrity during this micronucleation in PtK₁ cells. Observation of karyotypes obtained after 3 days of cell cycle restoration revealed that micronucleation did not affect chromosome integrity or the presence of each chromosome pair in the surviving cells. In early restoration cells, all the chromosomes included a centromere and were represented in the karyotype, but at variable rates. Furthermore, flow cytometry analysis of micronucleated cells, intermediate in DNA rate between control PtK₁ cells in g₁ and those in G₂/M phases, led us to consider the possibility of selective replication of some chromosomes during micronucleation. Using antibodies against the kinetochore proteins, we derived the presence of one centromeric region (1–2 spots) in the smallest micronuclei. Therefore, these data (karyotypes, number of chromosomes, DNA content and kinetochore proteins) seem to indicate that micronucleation does not induce chromosome damages or translocations. Micronuclei are a convenient tool for investigation of the role of the different chromosomes in the organization of the interphase nuclei.     

Quantitative Analysis of Centromeric FISH Spots during the Cell Cycle by Image Cytometry

Two-color fluorescence in situ hybridization (FISH) with chromosome enumeration DNA probes specific to chromosomes 7, 11, 17, and 18 was applied to CAL-51 breast cancer cells to examine whether the fluorescence intensity of FISH spots was associated with cell cycle progression. The fluorescence intensity of each FISH spot was quantitatively analyzed based on the cell cycle stage determined by image cytometry at the single-cell level. The spot intensity of cells in the G₂ phase was larger than that in the G_0/1 phase. This increased intensity was not seen during the early and mid S phases, whereas the cells in the late S phase showed significant increases in spot intensity, reaching the same level as that observed in the G₂ phase, indicating that alpha satellite DNA in the centromeric region was replicated in the late S phase. Thus, image cytometry can successfully detect small differences in the fluorescence intensities of centromeric spots of homologous chromosomes. This combinational image analysis of FISH spots and the cell cycle with cell image cytometry provides insights into new aspects of the cell cycle. This is the first report demonstrating that image cytometry can be used to analyze the fluorescence intensity of FISH signals during the cell cycle.     

Localization of chromosome regions in potoroo nuclei (<Emphasis Type="Italic"> Potorous tridactylus </Emphasis>Marsupialia: Potoroinae)

Chromosome paints of the rat kangaroo ( Aepyprymnus rufuscens, 2n =32) were used to define chromosome regions in the long nosed potoroo ( Potorous tridactylus, 2n =12 female, 13 male) karyotype and localize these regions in three-dimensionally preserved nuclei of the potoroo to test the hypothesis that marsupial chromosomes have a radial distribution. In human nuclei chromosomes are distributed in a proposed radial fashion. Gene-rich chromosomes in the human interphase nucleus are preferentially located in the central area while gene-poor chromosomes are found more at the periphery of the nucleus; this feature is conserved in primates and chicken. Chromosome ordering in nuclei of P. tridactylus is related to their size and centromere position. Its relationship with replication patterns in interphase nuclei and metaphase was studied. In addition it was observed that the nucleus was not a smooth entity but had projections occupied by specific chromosome regions. Edited by: R. Allshire     

Entry into Mitosis in Vertebrate Somatic Cells Is Guarded by a Chromosome Damage Checkpoint That Reverses the Cell Cycle When Triggered during Early but Not Late Prophase

When vertebrate somatic cells are selectively irradiated in the nucleus during late prophase (<30 min before nuclear envelope breakdown) they progress normally through mitosis even if they contain broken chromosomes. However, if early prophase nuclei are similarly irradiated, chromosome condensation is reversed and the cells return to interphase. Thus, the G₂ checkpoint that prevents entry into mitosis in response to nuclear damage ceases to function in late prophase. If one nucleus in a cell containing two early prophase nuclei is selectively irradiated, both return to interphase, and prophase cells that have been induced to returned to interphase retain a normal cytoplasmic microtubule complex. Thus, damage to an early prophase nucleus is converted into a signal that not only reverses the nuclear events of prophase, but this signal also enters the cytoplasm where it inhibits e.g., centrosome maturation and the formation of asters. Immunofluorescent analyses reveal that the irradiation-induced reversion of prophase is correlated with the dephosphorylation of histone H1, histone H3, and the MPM2 epitopes. Together, these data reveal that a checkpoint control exists in early but not late prophase in vertebrate cells that, when triggered, reverses the cell cycle by apparently downregulating existing cyclin-dependent kinase (CDK1) activity.     

Mitosis in flat PTK2-human hybrid cells

The fusion of G0 human fibroblasts with PTK₂ (Potorous tridactylis) cells resulted in the production of hybrid heterokaryotic cells which remained flat in cell division. These cells permitted studies of mitosis in living hybrid cells without the need for fixation and staining. The breakdown of nuclear envelopes during prophase in a hybrid heterokaryotic cell correlated with the onset of premature chromosome condensation (PCC) in other nuclei in the same cell. Nuclear morphology and autoradiography demonstrated that the nuclei exhibiting PCC were from the human parent cells. Observation of multinucleated PTK₂-human hybrids in the later stages of mitosis showed that these cells normally produced three daughters instead of the usual two. Electron microscopic examination of dividing hybrid cells showed that the number of daughter cells was not related to the number of centrioles. Hybrid cells normally were found to contain many centriolar duplexes although not all of these structures were associated with active poles in mitosis. Cells with as many as six centriolar duplexes were found in mitosis. The configuration of the chromosomes in metaphase was found to be a more accurate indication of the number of daughters produced by a single division than the number of centrioles. Chromosome elimination in hybrid cells could also be visualized in PTK₂-human hybrids. Lagging chromosomes were commonly observed during mitosis and were often trapped in the constricting midbody.     

The G1 period

In previous papers the existence of two cycles of chromosome condensation-decondensation per cell cycle was suggested based on experiments involving nuclear morphometry measurements of Feulgen-stained nuclei. This conclusion can be criticized since its assumption of a relationship between nuclear morphology and chromatin structure is derived from indirect evidence. In this paper, we report simultaneous measurements of nuclear area and nuclear fluorescence intensity on individual cells stained with the intercalating dye, acridine orange (AO). Using cells in various stages of G₁ and synchronized by two different methods, our results demonstrate a linear correlation between nuclear area and fluorescence intensity. They also indicate two cycles of chromatin condensation-decondensation during the G₁ period, as assayed by the number of chromatin primary, intercalating AO binding sites. Finally, they show that the first of these cycles involves a transition in early G₁ from a very small condensed nucleus (immediately after telephase) to a relatively large, dispersed nucleus that occurs abruptly.