Cell Cycle In Cultures Research Articles

Nuclear import of Xenopus egg extract components into cultured cells for reprogramming purposes: a case study on goldfish fin cells

Reprogramming of cultured cells using Xenopus egg extract involves controlling four major steps: plasma membrane permeabilization, egg factors import into the nucleus, membrane resealing, and cell proliferation. Using propidium iodide to assess plasma membrane permeability, we established that 90% of the cultured fin cells were permeabilized by digitonin without any cell losses. We showed that egg extract at metaphase II stage was essential to maintain nuclear import function in the permeabilized cells, as assessed with a fusion GFP protein carrying the nuclear import signal NLS. Moreover, the Xenopus-egg-specific Lamin B3 was detected in 87% of the cell nuclei, suggesting that other egg extract reprogramming factors of similar size could successfully enter the nucleus. Lamin B3 labelling was maintained in most cells recovered 24 h after membrane resealing with calcium, and cells successfully resumed cell cycle in culture. In contrast, permeabilized cells that were not treated with egg extract failed to proliferate in culture and died, implying that egg extract provided factor essential to the survival of those cells. To conclude, fish fin cells were successfully primed for treatment with reprogramming factors, and egg extract was shown to play a major role in their survival and recovery after permeabilization.

Circadian Clock Synchronization of the Cell Cycle in Zebrafish Occurs through a Gating Mechanism Rather Than a Period-phase Locking Process

Studies from a number of model systems have shown that the circadian clock controls expression of key cell cycle checkpoints, thus providing permissive or inhibitory windows in which specific cell cycle events can occur. However, a major question remains: Is the clock actually regulating the cell cycle through such a gating mechanism or, alternatively, is there a coupling process that controls the speed of cell cycle progression? Using our light-responsive zebrafish cell lines, we address this issue directly by synchronizing the cell cycle in culture simply by changing the entraining light-dark (LD) cycle in the incubator without the need for pharmacological intervention. Our results show that the cell cycle rapidly reentrains to a shifted LD cycle within 36 h, with changes in p21 expression and subsequent S phase timing occurring within the first few hours of resetting. Reentrainment of mitosis appears to lag S phase resetting by 1 circadian cycle. The range of entrainment of the zebrafish clock to differing LD cycles is large, from 16 to 32 hour periods. We exploited this feature to explore cell cycle entrainment at both the population and single cell levels. At the population level, cell cycle length is shortened or lengthened under corresponding T-cycles, suggesting that a 1:1 coupling mechanism is capable of either speeding up or slowing down the cell cycle. However, analysis at the single cell level reveals that this, in fact, is not true and that a gating mechanism is the fundamental method of timed cell cycle regulation in zebrafish. Cell cycle length at the single cell level is virtually unaltered with varying T-cycles.

Transcriptional Analysis of the Candida albicans Cell Cycle

We have examined the periodic expression of genes through the cell cycle in cultures of the human pathogenic fungus Candida albicans synchronized by mating pheromone treatment. Close to 500 genes show increased expression during the G1, S, G2, or M transitions of the C. albicans cell cycle. Comparisons of these C. albicans periodic genes with those already found in the budding and fission yeasts and in human cells reveal that of 2200 groups of homologous genes, close to 600 show periodicity in at least one organism, but only 11 are periodic in all four species. Overall, the C. albicans regulatory circuit most closely resembles that of Saccharomyces cerevisiae but contains a simplified structure. Although the majority of the C. albicans periodically regulated genes have homologues in the budding yeast, 20% (100 genes), most of which peak during the G1/S or M/G1 transitions, are unique to the pathogenic yeast.

Metaphase Chromosome Preparation from Cultured Peripheral Blood Cells

Chromosome preparations currently provide the only direct view of the genome as a whole. Although molecular methods allow a more detailed analysis of specific regions of the genome, the study of genetics is not complete without an appreciation of the metaphase cell. The stimulated T cell system described in this unit is the most widely used means of obtaining large numbers of mitotic cells for genetic analyses. Synchronization of the cell cycle in culture is described, combined with direct inhibition of chromosome condensation, to yield longer high-resolution prophase or prometaphase preparations. Such preparations are used for detailed analysis of microdeletions or subtle rearrangements, fine breakpoint analysis, and refined mapping. Microscope slide preparation of mitotic chromosomes from harvested cell culture suspensions is also explained in the support protocol.

Human CDK10 Gene Isoforms

The CDK10/PISSLRE gene has been shown to encode two different CDK-like putative kinases. The function(s) of the gene products are unknown, although a role at the G2/M transition has been suggested. We characterised two novel cDNAs. CDK10 mRNA quantity was not found to be correlated with cell proliferation status in HeLa or WI38 cell cultures or in human tissues. Relative levels of the four CDK10 isoforms were studied by RT-PCR, of which three were principally expressed. The two initially cloned isoforms predominated in human tissues, except in brain and muscle. Relative isoform levels did not vary during the cell cycle in culture, except when cells entered into the cell cycle. Finally, the predominant isoforms were shown to have different translation initiation sites and to have different subcellular distribution, due to an alternatively spliced nuclear localisation signal.

Molecular Cloning and Characterization of RGC-32, a Novel Gene Induced by Complement Activation in Oligodendrocytes

Sublytic complement activation on oligodendrocytes (OLG) down-regulates expression of myelin genes and induces cell cycle in culture. Differential display (DD) was used to search for new genes whose expression is altered in response to complement and that may be involved in cell cycle activation. DD bands showing either increased or decreased mRNA expression in response to complement were identified and designated Response Genes to Complement (RGC) 1-32. RGC-1 is identical with heat shock protein 105, RGC-2 with poly(ADP-ribose) polymerase, and RGC-10 with IP-10. A new gene, RGC-32, that encodes a protein of 137 amino acids was cloned. RGC-32 has no homology with other known proteins, and contains no motif that would indicate its function. In OLG, the mRNA expression was increased by complement activation and by terminal complement complex assembly. RGC-32 protein was localized in the cytoplasm and co-immunoprecipitated with cdc2 kinase. Overexpression of RGC-32 increased DNA synthesis in OLGxC6 glioma cell hybrids. These results suggest that RGC-32 may play a role in cell cycle activation.

Chromosome Preparation from Cultured Peripheral Blood Cells

AbstractChromosome preparations currently provide the only direct view of the genome as a whole. Although molecular methods allow a more detailed analysis of specific regions of the genome, the study of genetics is not complete without an appreciation of the metaphase cell. The stimulated T cell system described in this unit is the most widely used means of obtaining large numbers of mitotic cells for genetic analyses. Synchronization of the cell cycle in culture is described, combined with direct inhibition of chromosome condensation, to yield longer high‐resolution prophase or prometaphase preparations. Such preparations are used for detailed analysis of microdeletions or subtle rearrangements, fine breakpoint analysis, and refined mapping. Microscope slide preparation of mitotic chromosomes from harvested cell culture suspensions is also explained in the support protocol.

The age-associated increase in IFN-gamma synthesis by mouse CD8+ T cells correlates with shifts in the frequencies of cell subsets defined by membrane CD44, CD45RB, 3G11, and MEL-14 expression.

The mouse model system was used to evaluate age-associated changes in the subset composition and function of the splenic CD8+ T cell pool. In response to stimulation with plate-bound anti-CD3 epsilon mAb, CD8+ cells from old C57BL/6NNia mice produced greater levels of IFN-gamma than cells from young-adult controls. This age-associated difference was apparent at the levels of both IFN-gamma mRNA accumulation and cytokine release, and was established within the first major cell cycle in culture. In addition, the capacity to produce IFN-gamma appeared to increase gradually with age as evidenced by studies on CD8+ cells from intermediate aged mice. In contrast to these findings, the peak S-phase responses by stimulated CD8+ cells from old mice were significantly reduced relative to young-adult controls. Immunophenotypic analyses of membrane CD44, CD45RB, 3G11, and MEL-14 expression by splenic CD8+ cells from young-adult, intermediate-aged, and old mice revealed an age-associated decrease in the frequencies of cells that expressed low levels of CD44 and high levels of CD45RB, 3G11, and MEL-14, whereas the reciprocal phenotypes increased with age. The correlated analysis of all four subset markers identified a composite phenotype (CD44loCD45RBhiMEL-14hi3G11lo/hi) which, based on past functional studies, is a candidate phenotype for naive cells. This "naive" phenotype dominated the CD8+ cell pool of young-adult mice but decreased in frequency with age. In contrast to the CD44lo cell group, the CD44hi cell fraction, which is associated with preactivated/memory CD8+ cells, was found to be uniformly 3G11lo but expressed heterogeneous levels of CD45RB and MEL-14, perhaps defining multiple subsets within the memory population. All of these latter subsets increased in frequency with age. Finally, we found that when CD8+ cells were fractionated based on CD44 expression the capacity to release measurable levels of IFN-gamma segregated entirely with the CD44hi fraction, irrespective of donor age. Together, these findings support the hypothesis that aging is accompanied by dramatic shifts in the subset compositions of splenic CD8+ cell pools, which contribute significantly to their increased capacity to produce IFN-gamma at the population level.

The chronic effects of db-cAMP on cell cycle parameters and cell size in asynchronous culture of Chinese hamster ovary cells

N 6,O 2′-dibutyryl adenosine 3′,5′-cyclic-phosphate (db-cAMP) has been shown to convert Chinese hamster cells of ovarian origin (CHO-K 1) from compact, randomly oriented cells growing in multilayers to elongated fibroblast-like cells which grow in monolayers. This compound also has been reported to have a variety of effects on the cell cycle. Most such studies have employed synchronized cells to determine cell cycle effects, and consequently have been limited to the short-term effects of the compound. We have looked for chronic effects on the cell cycle in cultures exposed continuously to db-cAMP from the initiation of the cultures until they had reached or approached the plateau phase. This was done by combined autoradiography and Feulgen microspectrophotometry plus measurements of the protein content of mitotic cells to detect any influence on cell size. The overall results were that continuous exposure to db-cAMP had at most only minor effects on the cell cycle and cell size when the culture medium was renewed daily. Somewhat greater effects were found on plateau-phase cells in cultures in which the medium was not renewed. In this case fewer cells appeared to remain in the cell cycle in the cultures with db-cAMP. Comparison with our earlier results with Chinese hamster V79 cells led to the conclusions that cell cycle parameters and cell size at mitosis were less altered during culture growth in CHO cells, but that CHO cells seemed to be less able to maintain cells in the cell cycle in crowded cultures.

DNA synthesis and the cell cycle in cultures of normal and mutant ( [formula omitted]) embryonic mouse muscle cells

The relationship between cell fusion, DNA synthesis and the cell cycle in cultured embryonic normal and dysgenic ( mdg mdg ) mouse muscle cells has been determined by autoradiography. The experimental evidence shows that the homozygous mutant myotubes form by a process of cell fusion and that nuclei within the myotubes do not synthesize DNA or undergo mitotic or amitotic division. The duration of the total cell cycle and its component phases was statistically the same in 2-day normal and mutant ( mdg mdg ) myogenic cultures with the approximate values: T, 21.5 hr; G 1, 10.5 hr; S, 7.5 hr; and G 2, 2.5 hr. In both kinds of cultures, labeled nuclei appeared in myotubes 15–16 hr after mononucleated cells were exposed to [ 3H]thymidine, and the rate of incorporation of labeled nuclei into multinucleated muscle cells was comparable in control and dysgenic cultures. Thus, homozygous mdg mdg muscle cells in culture are similar to control cells with respect to their mechanism of myotube formation and the coordinate regulation of DNA synthesis and the cell cycle during myogenesis.

The duration of the cell cycle of Daucus carota L. in vivo and in vitro

The duration of the cell cycle in Daucus carota seedling root-tips was measured by scoring the fraction of labelled metaphases after pulse labelling with ( 3H-TdR). This gave a total cycle duration of 7.5 h almost equally divided between the component stages, G 1, S, G 2 and M. The duration of the cell cycle in established diploid and tetraploid suspension cultures grown in the presence of 2,4-D was measured by continuous labelling with 3H-TdR. This gave mean cell cycle times almost identical with the mean cell number doubling times (specific growth rates) obtained from cell number growth curves. Specific growth rates were then used as estimates of the mean cell cycle duration for diploid and tetraploid lines grown in the presence and absence of 2,4-D. As the continuous labelling experiments showed that 93% of cells could incorporate 3H-TdR, cell cycle phase durations were calculated from measurements of the proportions of cells in G 1, S, G 2 and M. These proportions were determined by a combination of autoradiography, microdensitometry and mitotic index determination on culture samples incubated for short periods with 3H-TdR. In the presence of 2,4-D, both diploid and tetraploid cultures had cell cycle times of 45–58 h. In the absence of 2,4-D the diploid culture actively produced embryo plantlets with a mean cell cycle time of 33 h, whereas the tetraploid culture showed a cell cycle time of 82 h and remained undifferentiated. The extension and variation of the cell cycle in culture relative to the root-tip meristem was due almost entirely to changes in the duration of G 1. However, except in the root-tip and the differentiating diploid culture, G 1 and G 2 occupied constant proportions of the cell cycle. S phase duration under all cultural conditions was similar to that found in the root-tips but mitotic duration was variably increased. Despite this variation in mitotic duration, mitotic phase proportions in culture were mostly similar to those of the root-tip.

Studies of Histone Methylation during the HeLa S-3 Cell Cycle

Abstract The rate and specificity of histone methylation were examined throughout the HeLa S-3 cell cycle in cultures synchronized by selective detachment in mitosis or by double thymidine block. (a) Of all the cellular fractions tested, only the 0.25 n H2SO4-soluble nuclear fraction contained proteins which appeared to be methylated at high rates. Within this fraction histone polypeptides f2a1 and f3 were methylated at the highest rates, f2b and f2a2 were methylated at lower rates and f1 polypeptides were not methylated at all. (b) Lysine residues in histone fractions f2a, f2b, and f3 were methylated throughout the cell cycle with maximum rates of methylation occurring in G2 phase and mitosis. Only histone f3 polypeptides contained significant amounts of methylated arginine and histidine residues. These residues were methylated at maximum rates during the S phase of the cell cycle. (c) Mitotic cells contained a higher percentage of methylated histone molecules than cells at other stages of the cell cycle and it was calculated that if there was only one methylation site in each of the molecules, from 80 to 100% of all f2a1, f2b, and f3 polypeptides were methylated. (d) The ratios of e, e-N-dimethyllysine to e-N-monomethyllysine in the histone fractions of the heteroploid malignant HeLa S-3 cell were about half the ratios of these amino acids reported found in the corresponding histones of normal cells. (e) The turnover rate of histone methyl groups was about 1 to 2% per hour depending upon the fraction.