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PubMed2004

Chromosomal aberrations and in vitro radiosensitivity: intra-individual versus inter-individual variability.

In order to assess the applicability of the micronucleus (MN) and G2 assays as biomarkers of in vitro radiosensitivity and cancer susceptibility, we investigated the inter- and intra-individual variation of these endpoints. For the MN assay unstimulated blood cultures from 57 healthy donors were exposed in vitro to 3.5 Gy Co gamma-rays and for the G2 assay PHA stimulated cultures were irradiated with a dose of 0.4 Gy Co gamma-rays in the G2 phase of the cell cycle. For 14 donors, 2-15 repeat samples were tested over a period of 3 years. The repeat experiments revealed that the intra-individual variability was not significantly different from the inter-individual variability for both G2 and MN assays. As the intra-individual variability determines the reproducibility of the assay, our results highlight the limitations of these endpoints in detecting reproducible differences in radiation sensitivity between individuals within a normal population. Due to the high intra-individual variability and no significant difference with the inter-individual variability found in our study we conclude that care has to be taken when results obtained with chromosomal aberration assays based on one blood sample are used to assess the individual radiosensitivity. Multiple blood sampling may be necessary to draw reliable conclusions. Although more validation studies on the reliability of the G2 and MN assay will be required before they can be used in a confident way as biomarkers of individual radiosensitivity or cancer susceptibility the assays are very valuable to examine population radiosensitivity and the relationship between radiosensitivity, cancer predisposition and genotype.

Adult↗
PubMed1999

Chromatid break rejoining and exchange aberration formation following gamma-ray exposure: analysis in G2 human fibroblasts by chemically induced premature chromosome condensation.

PURPOSE: To analyse the kinetics of chromatid break induction, rejoining, and misrejoining after y-irradiation in G2 phase human cells using premature chromosome condensation induced by calyculin A. MATERIALS AND METHODS: Human fibroblast AG1522 cells were irradiated with gamma-rays and chromosomes were then prematurely condensed by calyculin A. The number of chromatid breaks and chromatid exchanges in G2 chromosomes were scored, and fitted curves were calculated. RESULTS: Calyculin A induced premature chromosome condensation in cells immediately after irradiation. Kinetics of rejoining of chromatid breaks demonstrated two exponential components with rapid and slow time constants. Within 5 min after irradiation, the number of chromatid breaks fell rapidly to about one-half, then gradually decreased. Chromatid exchanges were formed very quickly, reaching a plateau within 20 min from exposure. CONCLUSIONS: Chemically induced premature chromosome condensation technique allows a simple, rapid and precise analysis of chromatid breakage and rejoining. The rapid kinetic component was particularly well characterized.

Cells, Cultured↗
PubMed2005

[Kinetic features of a white rat hepatocyte population under hormonal disbalance before and after partial hepatectomy].

Bilateral adrenalectomy, followed in 4 days by a partial hepatectomy, was performed using white rats weighing as much as 120-140 g. Under hormonal disbalance caused by bilateral adrenalectomy, the number of polyploid (4c, 4c x 2, and 8c) hepatocytes significantly increased, compared to that in non-operated control rats. Six hours after a partial hepatectomy, the share of highly ploid hepatocytes falls, being accompanied by a 9-fold increase in mitotic index. It is supposed that under hormonal disbalance condition, a partial hepatectomy may induce "early" mitoses in hepatocytes blocked in G2-phase of the cell cycle.

Adrenal Glands↗
PubMed1998

Comparison of routine flow cytometric DNA analysis of fresh tissues in two laboratories: effects of differences in preparation methods and background models of cell cycle calculation.

Routine flow cytometric DNA analysis was compared in two laboratories by using matched fresh-frozen breast cancer and soft tissue sarcoma biopsy specimens. Laboratory I applied the Vindelöv preparation method and an exponential background subtraction algorithm in the cell cycle calculation. Laboratory II used the Formalin-protease preparation technique and the sliced-nuclei background model. The results of the ploidy analysis showed good agreement between the two laboratories; however, the results of the cell cycle analysis showed considerable systematic differences between labs. Laboratory I obtained significantly lower values of S-phase fraction and higher values of G2-phase fraction than laboratory II. To explain these discrepancies, the effects of differences in the preparation methods and background subtraction algorithms were studied. The Vindelöv preparation method yielded higher debris and aggregation levels than the Formalin-protease technique and tended to give higher %S and %G2 values. When the two background models were used in the same histograms, the exponential background model tended to give %S values distinctly lower than and %G2 values almost identical to those obtained with the sliced-nuclei algorithm. The sum of these effects accounts for the observed inter-laboratory discrepancies. Different from the sliced-nuclei fit, the exponential background fit often did not accommodate to the original data in the <2c histogram region and resulted in a considerable inter-operator variability of %S calculation in histograms with <5% S. When aggregate correction was added to the sliced-nuclei algorithm, the differences between %S values in histograms from the two laboratories almost disappeared.

Algorithms↗
PubMed1997

Dynamics of cell cycle regulators: artifact-free analysis by recultivation of cells synchronized by centrifugal elutriation.

Studies on the molecular properties of cell cycle regulators in animal cells require cell preparations highly enriched in particular cell cycle phases. Centrifugal elutriation is frequently used to synchronize cells because this technique was thought to cause only minimal distortions in protein expression or metabolic functions. However, in primary chicken erythroblasts, we consistently observed artefacts in mitotic cyclin mRNA expression and p70 S6 kinase activity, which were clearly caused by the elutriation procedure. Therefore, we modified the standard protocol by reseeding various elutriated fractions into preconditioned medium, a process termed recultivation, and harvesting after an appropriate amount of time. This avoided the pleiotropic effects caused by stress and lack of growth factor supply during elutriation. Using this recultivation procedure, highly synchronous progression starting from any given cell cycle phase could be achieved for a variety of cell types, including primary, factor-dependent cells of hematopoietic origin. Mitotic cyclin expression and S6 kinase activity was found to be normal again in recultivated cultures, as opposed to elutriated ones. Finally, monitoring of mitosis-specific cyclin A degradation in recultivated G2 phase cells showed that recultivation provided an excellent tool to follow cells through M phase into G1 without the requirement for a chemical cell cycle block.

Animals↗
PubMed2000

The cell cycle genes cycA1;1 and cdc2Os-3 are coordinately regulated by gibberellin in planta.

Internodal growth of deepwater rice (Oryza sativa L.) can achieve rates of up to 10 mm h(-1). In submergence-induced plants, gibberellic acid activates the cell division cycle first at the G1 --> S phase transition with a subsequent increase in mitotic activity. The proteins cycA1;1 and cdc2Os-3 are the regulatory and catalytic subunits, respectively, of cyclin-dependent protein kinases (CDKs) which are central to cell cycle regulation. Both genes are regulated by gibberellic acid in a coordinate manner with transcripts accumulating in the G2 phase prior to the B2-type mitotic cyclins described previously (M. Sauter et al. 1995, Plant J 7: 623-632), suggesting a distinct role in regulating G2/M phase progression. Since cdc2Os-3 belongs to a group of CDKs that have no counterparts in animals, it may function in a plant-specific gibberellin-regulated cell division process.

Cell Cycle↗
PubMed2000

Microbiology. Arresting features of bacterial toxins.

Bacteria produce an arsenal of sophisticated toxins that disrupt the normal processes of the host cell, usually by modifying or inactivating host cell proteins. Now, as Coburn and Leong discuss in their Perspective, members of the cytolethal distending toxin (CDT) family have been identified as enzymes that attack DNA (and not protein) within the host cell (Lara-Tejero and Galán). By attacking DNA, perhaps during chromosomal replication, CDTs cause the host cell to halt in G2 phase of the cell cycle.

Animals↗
PubMed1994

The phenotype of a "Cdc2 kinase target site-deficient" mutant of oncoprotein 18 reveals a role of this protein in cell cycle control.

Oncoprotein 18 (Op18) is a cytosolic protein that is expressed in all proliferating cells. This phosphoprotein is up-regulated in a variety of human neoplasm, and phosphorylation of its two Cdc2 kinase target sites, Ser-25 and Ser-38, fluctuates dramatically during the cell cycle. We have investigated the potential role of the Cdc2 kinase-mediated phosphorylation of these two sites by expressing a "Cdc2 kinase target site-deficient" mutant of Op18 (Op18-S25,38A), and analyzed the phenotype on the level of cell cycle regulation. The result shows that induced expression of Op18-S25,38A results in rapid accumulation of cells in the G2 phase of the cell cycle. The block in G2 seems transient, since prolonged incubation was found to result in a large fraction of the transfected cells entering S phase in the absence of mitosis, i.e. endoreduplication. In addition, a fraction (30%) of the transfected cells was blocked in mitosis. Whereas the morphology of the G2 arrested cells appeared normal, expression of Op18-S25,38A caused a serious defect during mitotic chromosome segregation. Analyses of the mechanism behind the phenotype of Op18-S25,38A suggest an essential role for Op18 during cell division and that the mutant interferes with the function of the endogenous gene product.

Amino Acid Sequence↗
PubMed1988

Elongation of G1 phase by transient exposure of rat 3Y1 fibroblasts to caffeine during the previous and present generations.

When density-arrested rat 3Y1 fibroblasts were stimulated to enter S phase by seeding sparsely in fresh medium, caffeine inhibited this stimulation. When compared at the doses that gave the same levels of the inhibition of entry into S phase, caffeine inhibited protein synthesis to a far lesser extent than cycloheximide. This indicates that caffeine affects some event(s) specific to entry into S phase rather than general protein synthesis. When cells synchronized at early S phase were exposed to caffeine, progression of S and G2 phases was prolonged by only 1 h (from 6 h to 7 h). However, after removal of caffeine at mitosis, the G1 phase was prolonged for 5 h (from 11 h to 16 h). These results are consistent with our model that the initiation of S phase is regulated throughout the period between the adjacent S phases. When cells were incubated with normal medium containing serum during the S and G2 periods, a subsequent 6-h pulse exposure to caffeine caused prolongation of G1 phase for 7 h (from 11 h to 18 h). On the other hand, when cells were incubated in the absence of serum during these periods, the prolongation was only 2 h (from 16 h to 18 h). Similarly, entry into S phase was prolonged only 2 h, when a 6-h pulse exposure to caffeine was given immediately after release from density arrest or serum-deprivation arrest. These results indicate the involvement of the relaxation process, which is not affected by caffeine, when serum-deprived cells or density-arrested cells restore the process prerequisite for entry into S phase.

Animals↗
PubMed2002

Cmk2, a novel serine/threonine kinase in fission yeast.

The cmk2 gene of Schizosaccharomyces pombe encodes a 504 amino acid protein kinase with sequence homology with the calmodulin-dependent protein kinase family. The cmk2(+) gene is not essential for cell viability but overexpression of cmk2(+) blocks the cell cycle at G2 phase and this inhibition is cdc2-dependent. The Cmk2 is a cytoplasmic protein expressed in a cell cycle-dependent manner, peaking at the G1/S boundary. Overexpression of Cmk2 suppresses fission yeast DNA replication checkpoint defects but not DNA damage checkpoint defects, suggesting that the G2 cell cycle arrest mediated by high levels of Cmk2 provides sufficient time to correct DNA replication alterations.

Amino Acid Sequence↗
PubMed2005

The Ste20-like kinase SLK is required for cell cycle progression through G2.

We have previously shown that the Ste20-like kinase SLK is a microtubule-associated protein that can regulate actin reorganization during cell adhesion and spreading (Wagner, S., Flood, T. A., O'Reilly, P., Hume, K., and Sabourin, L. A. (2002) J. Biol. Chem. 277, 37685-37692). Because of its association with the microtubule network, we investigated whether SLK plays a role in cell cycle progression, a process that requires microtubule dynamics during mitosis. Consistent with microtubule association in exponentially growing cells, our results showed that SLK co-localizes with the mitotic spindle in cells undergoing mitosis. Expression of a kinase-inactive mutant or SLK small interfering RNAs inhibited cell proliferation and resulted in an accumulation of quiescent cells stimulated to re-enter the cell cycle in the G2 phase. Cultures expressing the mutant SLK displayed a normal pattern of cyclin D, E, and B expression but failed to down-regulate cyclin A levels, suggesting that they cannot proceed through M phase. In addition, these cultures displayed low levels of both phospho-H3 and active p34/cdc2 kinase. Overexpression of active SLK resulted in ectopic spindle assembly and the induction of cell cycle re-entry of Xenopus oocytes, suggesting that SLK is required for progression through G2 upstream of H1 kinase activation.

Animals↗
PubMed1995

The RCK1 and RCK2 protein kinase genes from Saccharomyces cerevisiae suppress cell cycle checkpoint mutations in Schizosaccharomyces pombe.

The protein kinase-encoding genes RCK1 and RCK2 from Saccharomyces cerevisiae have been identified as suppressors of Schizosaccharomyces pombe cell cycle checkpoint mutations. Upon expression of these genes, radiation resistance is partially restored in S. pombe mutants with checkpoint deficiencies, but not in mutants with DNA repair defects. Some checkpoint mutants are sensitive to the DNA synthesis inhibitor hydroxyurea, and this sensitivity is also suppressed by RCK1 and RCK2. The degree of suppression can be modulated by varying expression levels. Expression of RCK1 or RCK2 in S. pombe causes cell elongation and decelerated growth. Cells expressing these genes have a single nucleus and a 2n DNA content. We conclude that these genes act in S. pombe to prolong the G2 phase of the cell cycle.

Base Sequence↗
PubMed2004

Effects of HMGB-1 overexpression on cell-cycle progression in MCF-7 cells.

High mobility group-1 (HMGB-1) enhances the DNA interactions and possesses a transcriptional activation potential for several families of sequence-specific transcriptional activators. In order to examine the effect of HMGB-1 on the cell cycle progression in MCF-7 cells, the HMGB-1 expression vector was transfected into synchronized MCF-7 cells, and the effect of HMGB-1 overexpression on the cell cycle was examined. The HMGB-1 protein level in the transfected cells increased 4.87-fold compared to the non-transfected cells. There were few changes in the cell cycle phase distribution after HMGB-1 overexpression in the MCF-7 cells. Following the estrogen treatment, the cell cycle progressed in both the HMGB-1 overexpressed MCF-7 and the mock-treated cells. However, a larger proportion of HMGB-1 overexpressing MCF-7 cells progressed to the either S or G2 phase than the mock-treated cells. The mRNA levels of the cell cycle regulators changed after being treated with estrogen in both the HMGB-1 overexpressing MCF-7 and the mock-treated cells, but the changes in the expression level of the cell cycle regulator genes were more prominent in the HMGB-1 overexpressing MCF-7 cells than in the mock-treated cells. In conclusion, HMGB-1 overexpression itself does not alter the MCF-7 cell cycle progression, but the addition of estrogen to the HMGB-1 overexpressing MCF-7 cells appears to accelerate the cell cycle progression.

Blotting, Western↗
PubMed1996

Vpr-induced cell cycle arrest is conserved among primate lentiviruses.

We previously reported that expression of human immunodeficiency virus type 1 strain NL4-3 (HIV-1(NL4-3))vpr causes cells to arrest in the G2 phase of the cell cycle. We examined the induction of cell cycle arrest by other HIV-1 isolates and by primary lentiviruses other than HIV-1. We demonstrate that the vpr genes from tissue culture-adapted or primary isolates of HIV-1 are capable of inducing G2 arrest. In addition, we demonstrate that induction of cell cycle arrest is a conserved function of members of two other groups of primate lentiviruses, HIV-2/simian immunodeficiency virus strain sm (SIVsm)/SIVmac and SIVagm. vpr from HIV-1, HIV-2, and SIVmac induced cell cycle arrest when transfected in human (HeLa) and monkey (CV-1) cells. vpx from HIV-2 and SIVmac did not induce detectable cell cycle arrest in either cell type, and SIVagm vpx was capable of inducing arrest in CV-1 but not HeLa cells. These results indicate that induction of cell cycle perturbation is a general property of lentiviruses that infect primates. The conservation of this viral function throughout evolution suggests that it plays a key role in virus-host relationships, and elucidation of its mechanism may reveal important clues about pathology induced by primary lentiviruses.

Alleles↗
PubMedDate not supplied

Confocal spectral imaging analysis of intracellular interactions of mitoxantrone at different phases of the cell cycle.

It is suggested that the cytotoxicity of anticancer agent mitoxantrone (MITOX) is related to a complex combination of molecular interactions which lead to slowing of S phase traverse and arresting of cells in G2 phase of the cell cycle or even to an apoptosis at high concentration of MITOX. Here intracellular molecular interactions of MITOX were visualised and studied using the confocal spectral imaging technique in synchronised K562 cells. Localisation, quantitative distributions of MITOX in the polar environment, MITOX bound to hydrophobic cellular structures (MITOXphob), nucleic acid-related complexes of MITOX (MITOXNA) and relative distributions of naphthoquinoxaline (NQX) metabolite and intrinsic cellular fluorescence of porphyrins were measured within cytoplasmic and nuclear compartments (chromosomes) of the G2, S, and M cells treated with 10 or 2 microM of MITOX for 1 hour. Colocalisation of MITOX, NQX metabolite and sites of intrinsic cellular fluorescence indicates an accumulation of MITOX within or near mitochondria. One may suppose that due to high concentration MITOX can compete with natural substrates for binding to the enzymes thus affecting the normal functioning of a mitochondria. A remarkable redistribution of MITOX and its complexes occurs in the M cells. In particular, a prominent amount of MITOX is associated with the surface of chromatids but not with the cytoplasmic structures in M cells. At the present time the exact location of the sites of MITOX accumulation in the M cells is not known. It is thought to be some cytoskeleton/microtubule structures associated directly with the chromosomes. Selective labelling of particular cytoskeleton structures and/or proteins in MITOX treated cells is in the progress now and the question will be addressed using the CSI technique.

Antineoplastic Agents↗
PubMed1991

[The parameters of tumor cell proliferation as a prognostic factor in lymphosarcomas].

Cytophotometry of DNA was used to measure cell fractions in the S + G2 phases of the mitotic cycle in imprints of the involved lymph nodes and/or smears of the cells isolated from archival paraffin blocks of 60 patients with lymphosarcoma. Parallel studies have shown that both cell sources permit obtaining similar results (r = 0.98, p less than 0.001). The mean S + G2 values increased with the increment of the histological degree of malignancy, amounting to 1.6 +/- 0.4, 3.4 +/- 0.5 and 7.4 +/- 2.7% for patients with low, intermediate and high degrees, respectively. However, the statistically significant differences in accordance with Wilcoxon's criterion were revealed but for the second and third groups. To estimate the independent prognostic importance of the S + G2 parameter, all the patients were distributed into 2 groups. The first group included patients, in whom the magnitude of S + G2 did not surpass 3%, whereas the second one was made up of patients with the S + G2 values exceeding 3%. In the second group, the mean survival reached 13.8 +/- 4.0 months. At present 45% (9 out of 20) are still alive. In the first group, the mean survival was 25.2 +/- 3.8 months, with 70% of the patients being alive (28 out of 40). The significance of the differences is very high: p less than 0.001. Thus, the S + G2 parameter turned out to be reversely dependent on the survival of lymphosarcoma patients.

Adult↗
PubMed2004

Inhibition of proliferation and survival of melanoma cells by adenoviral-mediated expression of dominant negative fibroblast growth factor receptor.

Melanoma is the most deadly cutaneous malignancy; its incidence has risen significantly over the last 30 years and there is no effective treatment for advanced melanoma. Autocrine expression of fibroblast growth factors (FGFs) is a common event in malignant melanoma. We therefore sought to inhibit the proliferation and survival of melanoma cells as an approach to melanoma therapy by disrupting FGF signalling via adenoviral-mediated expression of a dominant negative FGF receptor. We tested three melanoma cell lines (A375, IIB and UCD) that express FGF2 and respond to exogenous FGF2 with increased proliferation. In all three cell lines, there was a significant decrease in net proliferation, and in two (A375 and UCD) an increase in cell death, associated with expression of the dominant negative FGF receptor. In these two cell lines, expression of the dominant negative FGF receptor resulted in the accumulation of p21 (WAF1/cip1), decreased cell division cycle-2 (cdc2) kinase activity, an increase in the percentage of cells in the G2 phase of the cell cycle, and inhibition of cytokinesis, with accumulation of binucleated cells. These studies reveal that inhibition of FGF signalling can markedly decrease the net proliferation of melanoma cells by a novel mechanism involving a decrease in cdc2 kinase activity, an increase in p21 expression, inhibition of G2 progression, inhibition of cytokinesis and increased cell death. In contrast, disruption of FGF signalling had only slight effects on normal melanocytes. Disruption of FGF signalling via the expression of the dominant negative FGF receptor is, therefore, a potential therapeutic approach in human melanoma.

Adenoviridae↗
PubMed1995

Requirement for Src family protein tyrosine kinases in G2 for fibroblast cell division.

The protein tyrosine kinase c-Src is transiently activated at the transition from the G2 phase to mitosis in the cell cycle of mammalian fibroblasts. Fyn and Yes, the other members of the Src family present in fibroblasts, were also found to be activated at mitosis. In cells microinjected with a neutralizing antibody specific for Src, Fyn, and Yes (anti-cst.1) during G2, cell division was inhibited by 75 percent. The block occurred before nuclear envelope breakdown. Antibodies specific for phosphatidylinositol-3 kinase alpha and phospholipase C-gamma 1 had no effect. Microinjection of the Src homology 2 (SH2) domain of Fyn was also inhibitory. Functional redundancy between members of the Src family was observed; a Src-specific antibody had no effect in NIH 3T3 cells but inhibited cell division in fibroblasts in which the only functional Src family kinase was Src itself. Thus, Src family kinases and proteins associating with their SH2 domains are required for entry into mitosis.

3T3 Cells↗