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Results for “Resting Phase, Cell Cycle”

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PubMed1997

Amount of the two major Ag-NOR proteins, nucleolin, and protein B23 is cell-cycle dependent.

To know the biological basis allowing the use of Ag-NOR protein expression as proliferation marker in human malignancies, the relationship between cell cycle and amount of Ag-NOR protein was analyzed. The quantification of the two major Ag-NOR proteins, nucleolin and protein B23, was performed in exponentially growing, serum-deprived, and cell-cycle stimulated cells. Expression of nucleolin was low in serum-deprived cells and increased mostly in S phase during cell-cycle stimulation. Conversely, expression of protein B23 was slightly repressed in serum-deprived cells, and increased progressively until G2 phase during cell-cycle stimulation. The accumulation of nucleolin and protein B23 in G2 compared to G1 was demonstrated using sorted phase-specific cells. In G0, cells sorted according to their very low RNA content, and the amount of Ag-NOR proteins was half of that found in G1 cells, nucleolin being only weakly detectable. Therefore, the expression of nucleolin increased between G0-G1 and G1-S phases. These data support the hypothesis that quantification of Ag-NOR proteins is an estimation of the percentage of cells in each cell cycle phase because their amount is high in S-G2 and low in G1 phases.

Animals↗

PubMed1995

Induction of gadd153 gene in G0-specific ts mutant cells at nonpermissive temperature.

tsJT60 is a temperature-sensitive (ts) mutant of the cell cycle isolated from a rat cell line. It does not enter the cell cycle from the G0 phase at a nonpermissive temperature (40 degrees C) but proliferates normally in log phase at both permissive (34 degrees C) and nonpermissive temperatures (G0-specific ts mutant). Here we examined whether tsJT60 fails to downregulate growth arrest-specific genes after growth stimulation from G0 phase at 40 degrees C. Among growth arrest-specific genes examined, an expression of gadd153, which decreased after serum stimulation at 34 degrees C as expected, increased markedly at 40 degrees C. It is plausible to assume that induction of gadd153 at 40 degrees C is correlated with inability of tsJT60 to enter S phase from G0 phase at 40 degrees C, because it also occurred in tsJT366 (another ts mutant clone in the same complementation group) but did not occur in parental 3Y1 and in other ts mutant lines of different complementation groups. However, gadd153 was also induced after the temperature upshift in tsJT60 cells growing in log phase or progressing S phase.

Animals↗

PubMed2004

New method for the analysis of cell cycle-specific apoptosis.

BACKGROUND: In this study, a new method for the analysis of cell cycle specificity of apoptosis was designed by using a modified annexin V and propidium iodide (API) method. METHODS: Cells of the human promyelocytic HL-60 line treated with camptothecin (CPT) or ultraviolet light (UV) were labeled with fluorescein isothiocyanate-conjugated annexin V and prefixed with 1% methanol-free formaldehyde on ice, and their DNA was stained stoichiometrically with propidium iodide in the presence of digitonin. Cellular green and red fluorescences were measured by flow cytometry. RESULTS: Cell cycle specificity of apoptosis obtained by the API method and those analyzed for the presence of DNA strand breaks by using terminal deoxynucleotidyl transferase (TdT) assay were similar: CPT- or UV-induced apoptosis preferentially in S- or G1-phase cells, respectively. When the internucleosomal DNA degradation was prevented by the serine protease inhibitor N-tosyl-L-phenylalanine chloromethyl ketone, apoptotic cells could not be detected by the TdT assay but were identified by the API method. CONCLUSIONS: The API method, similar to the TdT assay, accurately detects the cell cycle phase specificity of apoptosis. Also, the API method appears to detect earlier stages of apoptosis than the TdT assay.

Annexin A5↗

PubMed2000

SLG1 plays a role during G1 in the decision to enter or exit the cell cycle.

Saccharomyces cerevisiae cells decide to divide during G1. If nutrients are abundant, cells pass through START and coordinately undergo DNA replication, bud emergence, and spindle pole body duplication. Phenotypic analysis of the slg1delta mutant revealed that this mutation uncouples post-START events. At the nonpermissive temperature, slg1delta cells that have undergone bud emergence but not DNA replication or SPB duplication accumulate. Furthermore, while wild-type cells arrest in GO when starved, the slg1delta mutant fails to arrest at this point; instead, cells with small buds accumulate. The slg1delta mutation displayed genetic interactions with cdc34, which encodes a regulator of exit from G1. This is consistent with a role of SLG1 in G1 regulation. Epitope-tagged Slg1p cofractionated with the plasma membrane, suggesting that Slglp may function by integrating external cues and relaying them to the interior of the cell. We propose that SLG1 plays a regulatory role in bud emergence or stationary phase.

Base Sequence↗

PubMed2004

Anti-proliferation effect of 5,5-diphenyl-2-thiohydantoin (DPTH) in human vascular endothelial cells.

The aim of this study was to examine the anti-proliferation effect of 5,5-diphenyl-2-thiohydantoin (DPTH), an analogue of antiepileptic drug phenytoin (5,5-diphenylhydantoin), on human umbilical vein endothelial cells (HUVEC) and its possible molecular mechanism underlying. Here we demonstrated that DPTH at a range of concentrations (12.5-50 microM) dose- and time-dependently inhibited DNA synthesis and decreased cell number in cultured HUVEC, but not human fibroblasts. DPTH was not cytotoxic at these concentrations. [3H]Thymidine incorporation and flow cytometry analyses demonstrated that treatment of HUVEC with DPTH arrested the cell at the G0/G1 phase of the cell cycle. Western blot analysis revealed that the protein level of p21 increased after DPTH treated. In contrast, the protein levels of p27, p53, cyclins A, D1, D3 and E, cyclin-dependent kinase (CDK)2, and CDK4 in HUVEC were not changed significantly after DPTH treatment. Immunoprecipitation showed that the formations of the CDK2-p21 and CDK4-p21 complex, but not the CDK2-p27 and CDK4-p27 complex, were increased in the DPTH-treated HUVEC. Kinase assay further demonstrated that both CDK2 and CDK4 kinase activities were decreased in the DPTH-treated HUVEC. Pretreatment of HUVEC with a p21 antisense oligonucleotide reversed the DPTH-induced inhibition of [3H]thymidine incorporation into HUVEC. In conclusion, these data suggest that DPTH inhibits HUVEC proliferation by increasing the level of p21 protein, which in turn inhibits CDK2 and CDK4 kinase activities, and finally interrupts the cell cycle. The findings from the present study suggest that DPTH might have the potential to inhibit the occurrence of angiogenesis.

Cell Cycle↗

PubMed2003

Coordination between donor cell type and cell cycle stage improves nuclear cloning efficiency in cattle.

Several studies have shown that both quiescent and proliferating somatic donor cells can be fully reprogrammed after nuclear transfer (NT) and result in viable offspring. So far, however, no comparative study has conclusively demonstrated the relative importance of donor cell cycle stage on nuclear cloning efficiency. Here, we compare two different types of bovine fetal fibroblasts (BFFs) that were synchronized in G(0), G(1), and different phases within G(1). We show that for non-transgenic (non-TG) fibroblasts, serum starvation into G(0) results in a significantly higher percentage of viable calves at term than synchronization in early G(1) or late G(1). For transgenic fibroblasts, however, cells selected in G(1) show significantly higher development to calves at term and higher post-natal survival to weaning than cells in G(0). This suggests that it may be necessary to coordinate donor cell type and cell cycle stage to maximize overall cloning efficiency.

Animals↗

PubMed2002

Regulation of Ras signaling by the cell cycle.

It is well known that upregulation of Ras activity can promote cell-cycle progression. Now recent studies indicate that a reciprocal relationship also exists; that is, the consequences of Ras signaling are dependent upon cell-cycle position. In quiescent cells stimulated with growth factors, one Ras effector, phosphatidylinositol-3-kinase, is activated twice as cells transition from G(0) into G(1) phase, and then later in G(1) phase. It is only during the later stages of G(1) phase that PI3K activity promotes entry into S-phase. In cycling cells, Ras activity is enhanced throughout the cell cycle, but is able to stimulate cyclin D1 elevation only during G(2) phase.

Animals↗

PubMed1992

[Preliminary studies of the relationship between micronucleus formation and cell cycle. IV. Micronucleus formation induced by chemical mutagens at G0, G1, S and G2 phases of human lymphocytes].

In this paper the authors studied quantitatively the micronucleus formation at various phases of interphase in human lymphocytes induced by chemical mutagens by means of control of cell culture intervals, autoradiography and block of metaphase cells etc. The results show that mutagenic anti-tumor drugs: bimolane (treatment in vitro) and thio-tepa etc. (treatment in vivo) can induce micronucleus formation at various phases of interphase in lymphocytes. The frequency of micronucleus (MNF) induced at G1 phase is significantly higher than that at G0 and G2 phase. The MNF at S phase of cells is obviously lower than at G1. This result suggests that most of the micronucleated cells at G1 phase do not enter S phase and micronucleus rarely form at S phase of cells.

Adolescent↗

PubMed1996

Cell cycle-specific behavior of erythropoietin.

The murine erythropoietin-dependent erythroleukemia cell line, HCD-57, was employed to study the cell cycle-specific behavior of erythropoietin. Cell cycle duration for HCD-57 cells was approximately 12 hours and was uninfluenced by erythropoietin. Populations of HCD-57 cells synchronized in G1 by centrifugal elutriation were able to pass through one complete cell cycle in the absence of erythropoietin but, thereafter, arrested in G1 as identified by propidium iodide staining and flow cytometry. Analysis of cell cycle behavior using the metachromic dye acridine orange, however, revealed that HCD-57 cells pass through a G0 cell cycle phase and, like serum-deprived 3T3 cells, actually arrest in G0 when deprived of erythropoietin. Expression of the cell cycle regulatory protein p34cdc2 was invariant throughout the cell cycle in HCD-57 cells. p34cdc2 was constitutively phosphorylated in G0 cells, and this effect was not modified by erythropoietin. Erythropoietin receptor distribution was log normal in HCD-57 cells in each phase of the cell cycle. The affinity of these surface receptors for erythropoietin was essentially invariant throughout the cell cycle, but receptor expression was upregulated in G2M cells as compared with cells in G1 or S phase. Taken together, these data indicate that erythropoietin has an important role in the G0-G1 to S phase transition but, based on receptor expression, is involved in other phases of the cell cycle as well.

Animals↗

PubMed1997

Functional heterogeneity of human CD34(+) cells isolated in subcompartments of the G0 /G1 phase of the cell cycle.

Using simultaneous Hoechst 33342 (Hst) and Pyronin Y (PY) staining for determination of DNA and RNA content, respectively, human CD34(+) cells were isolated in subcompartments of the G0 /G1 phase of the cell cycle by flow cytometric cell sorting. In both bone marrow (BM) and mobilized peripheral blood (MPB) CD34(+) cells, primitive long-term hematopoietic culture-initiating cell (LTHC-IC) activity was higher in CD34(+) cells isolated in G0 (G0CD34(+) cells) than in those residing in G1 (G1CD34(+) cells). However, as MPB CD34(+) cells displayed a more homogeneous cell-cycle status within the G0 /G1 phase and a relative absence of cells in late G1 , DNA/RNA fractionation was less effective in segregating LTHC-IC in MPB than in BM. BM CD34(+) cells belonging to four subcompartments of increasing RNA content within the G0 /G1 phase were evaluated in functional assays. The persistence of CD34 expression in suspension culture was inversely correlated with the initial RNA content of test cells. Multipotential progenitors were present in G0 or early G1 subcompartments, while lineage-restricted granulomonocytic progenitors were more abundant in late G1 . In vitro hematopoiesis was maintained for up to 6 weeks with G0CD34(+) cells, whereas production of clonogenic progenitors was more limited in cultures initiated with G1CD34(+) cells. To test the hypothesis that primitive LTHC-ICs would reenter a state of relative quiescence after in vitro division, BM CD34(+) cells proliferating in ex vivo cultures were identified from their quiescent counterparts by a relative loss of membrane intercalating dye PKH2, and were further fractionated with Hst and PY. The same functional hierarchy was documented within the PKH2(dim) population whereby LTHC-IC frequency was higher for CD34(+) cells reselected in G0 after in vitro division than for CD34(+) cells reisolated in G1 or in S/G2 + M. However, the highest LTHC-IC frequency was found in quiescent PKH2(bright) CD34(+) cells. Together, these results support the concept that cells with distinct hematopoietic capabilities follow different pathways during the G0 /G1 phase of the cell cycle both in vivo and during ex vivo culture.

Antigens, CD34↗

PubMed1996

Effects of cell cycle dependent histone H1 phosphorylation on chromatin structure and chromatin replication.

We have reconstituted salt-treated SV40 minichromosomes with differentially phosphorylated forms of histone H1 extracted from either G0-, S- or M-phase cells. Sedimentation studies revealed a clear difference between minichromosomes reconstituted with S-phase histone H1 compared with histone H1 from G0- or M-phase cells, indicating that the phosphorylation state of histone H1 has a direct effect on chromatin structure. Using reconstituted minichromosomes as substrate in the SV40 in vitro replication system, we measured a higher replication efficiency for SV40 minichromosomes reconstituted with S-phase histone H1 compared with G0- or M-phase histone H1. These data indicate that the chromatin structure induced by the phosphorylation of histone H1 influences the replication efficiency of SV40 minichromosomes in vitro.

Animals↗

PubMed1991

Adaptive response to chromosome damage in cultured human lymphocytes primed with low doses of X-rays.

Human lymphocytes exposed to 0.02 Gy of X-rays in the G1 but not the G0 phase became less susceptible to the induction of chromosome aberrations of the chromosome type by subsequent exposure to 3 Gy of X-rays. The induction of chromatid-type aberrations was not affected by the pretreatment with the priming dose. The expression of this adaptive-type response was transitory, being maximum at 5 h, and disappeared at 9 h after the initial low-dose exposure. Cell-cycle analysis excluded the possibility of a spurious consequence of differential cell-cycle progression.

Adaptation, Physiological↗

PubMed2001

Expression of topoisomerase IIalpha in the G0/G1 cell cycle phase of fresh leukemic cells.

Topoisomerase IIalpha (topo IIalpha) is the target enzyme for several antineoplastic drugs. Correlation between low expression of topo IIalpha and drug resistance has been shown in vitro, but there is limited evidence of a correlation to initial response to treatment or to overall prognosis. Normal cells express topo IIalpha in S/G2/M phase of the cell cycle but not in G0/G1 phase. However, some data suggest that topo IIalpha could be expressed in G0/G1 phase in malignant cells. We have investigated the expression of topo IIalpha in leukemic cells from 25 patients with acute leukemia by flow cytometry, separating cells of different cell cycle phases. We demonstrated that 9/25 samples showed >50% positive cells in G0/G1, and another five samples showed >20%. This finding could possibly provide an explanation to previous difficulties in correlating topo IIalpha expression with clinical outcome. Six of eight patients, where >20% of the cells in G0/G1 were positive for topo IIalpha, entered CR, compared to one of five patients with <20% topo IIalpha positive cells in G0/G1. We suggest that topo IIalpha expression in G0/G1 in leukemic cells may be of predictive value for clinical response to cytostatic drugs.

Acute Disease↗

PubMed2000

The APC-hDLG complex negatively regulates cell cycle progression from the G0/G1 to S phase.

The adenomatous polyposis coli (APC) gene is mutated in familial adenomatous polyposis and in many sporadic colorectal tumors. The carboxyl-terminal S/TXV motif of the APC gene product interacts with the PDZ domain of hDLG, the human homolog of the Drosophila lethal (1) discs larige-1 (dlg) tumor suppressor. In the present study, we found that overexpression of hDLG suppresses cell proliferation by blocking cell cycle progression from the G0/G1 to S phase. This inhibition of cell cycle progression was abolished when the PDZ, SH3 or guanylate kinase-like domain of hDLG was mutated. Moreover, overexpression of these mutant hDLGs partially interfered with the cell cycle blocking activity of APC. Consistent with this result, mutant APC lacking the S/TXV motif exhibited weaker cell cycle blocking activity than the intact APC. These results suggest that APC-hDLG complex formation plays an important role in transducing the APC cell cycle blocking signal.

3T3 Cells↗

PubMed1996

The accumulation of an E2F-p130 transcriptional repressor distinguishes a G0 cell state from a G1 cell state.

Previous studies have demonstrated cell cycle-dependent specificities in the interactions of E2F proteins with Rb family members. We now show that the formation of an E2F-p130 complex is unique to cells in a quiescent, G0 state. The E2F-p130 complex does not reform when cells reenter a proliferative state and cycle through G1. The presence of an E2F-p130 complex in quiescent cells coincides with the E2F-mediated repression of transcription of the E2F1 gene, and we show that the E2F sites in the E2F1 promoter are important as cells enter quiescence but play no apparent role in cycling cells. In addition, the decay of the E2F-p130 complex as cells reenter the cell cycle requires the action of G1 cyclin-dependent kinase activity. We conclude that the accumulation of the E2F-p130 complex in quiescent cells provides a negative control of certain key target genes and defines a functional distinction between these G0 cells and cells that exist transiently in G1.

3T3 Cells↗

PubMed2004

A novel approach in the treatment of neuroendocrine gastrointestinal tumors: additive antiproliferative effects of interferon-gamma and meta-iodobenzylguanidine.

BACKGROUND: Therapeutic options to effectively inhibit growth and spread of neuroendocrine gastrointestinal tumors are still limited. As both meta-iodobenzylguanidine (MIBG) and interferon-gamma (IFNgamma) cause antineoplastic effects in neuroendocrine gastrointestinal tumor cells, we investigated the antiproliferative effects of the combination of IFNgamma and non-radiolabeled MIBG in neuroendocrine gut STC-1 and pancreatic carcinoid BON tumor cells. METHODS AND RESULTS: IFNgamma receptors were expressed in both models. IFNgamma dose- and time-dependently inhibited the growth of both STC-1 and of BON tumor cells with IC50-values of 95 +/- 15 U/ml and 135 +/- 10 U/ml, respectively. Above 10 U/ml IFNgamma induced apoptosis-specific caspase-3 activity in a time-dependent manner in either cell line and caused a dose-dependent arrest in the S-phase of the cell cycle. Furthermore, IFNgamma induced cytotoxic effects in NE tumor cells. The NE tumor-targeted drug MIBG is selectively taken up via norepinephrine transporters, thereby specifically inhibiting growth in NE tumor cells. Intriguingly, IFNgamma treatment induced an upregulation of norepinephrine transporter expression in neuroendocrine tumors cells, as determined by semi-quantitative RT-PCR. Co-application of sub-IC50 concentrations of IFNgamma and MIBG led to additive growth inhibitory effects, which were mainly due to increased cytotoxicity and S-phase arrest of the cell cycle. CONCLUSION: Our data show that IFNgamma exerts antiproliferative effects on neuroendocrine gastrointestinal tumor cells by inducing cell cycle arrest, apoptosis and cytotoxicity. The combination of IFNgamma with the NE tumor-targeted agent MIBG leads to effective growth control at reduced doses of either drug. Thus, the administration of IFNgamma alone and more so, in combination with MIBG, is a promising novel approach in the treatment of neuroendocrine gastrointestinal tumors.

3-Iodobenzylguanidine↗

PubMed1994

Cell kinetic characterization of growth arrest in cultured human keratinocytes.

In this study we have performed a cell kinetic characterization of growth and growth arrest of keratinocytes derived from normal human skin. Proliferative activity of the cell cultures was analysed with a flow cytometric technique, measuring relative DNA content and iododeoxyuridine (IdUrd) incorporation simultaneously. Normal human keratinocytes were grown in keratinocyte growth medium (KGM) and growth arrest was induced by using either keratinocyte basal medium (KBM) or KGM supplemented with TGF-beta 1. It was found that human keratinocytes grown in KGM plus TGF-beta 1 were growth-arrested within 52 hours. The rate of IdUrd incorporation into DNA decreased by more than 95% after 52 hours and paralleled the decrease of cells in S-phase. Within 52 hours after addition of TGF-beta 1, 79% of the growth-arrested cells were in the G0/G1-phase of the cell cycle, a situation that approaches that of the normal epidermis. Growth arrest of human keratinocytes in KBM showed a similar decrease in the rate of IdUrd incorporation. However, the decrease in IdUrd incorporation was not reflected in a decrease in cells in S-phase, suggesting that the cells were blocked in G0/G1, S or G2/M-phase rather than selectively in the physiological growth arrest state of G0/G1. Secondly, we investigated the kinetics of the cells when they were restimulated after growth arrest. We found that after termination of the growth arrest in KGM supplemented with TGF-beta 1 the cells require 6 to 8 hours to initiate DNA synthesis, with a continued decrease in the G0/G1 population, suggesting that the cells are recruited as a cohort. After growth arrest induced by KBM, cells also require 6 to 8 hours in KGM to initiate DNA synthesis, but the cells are not recruited as a cohort. We conclude that growth arrest induced by TGF-beta 1 is the preferred system in which to study induction of keratinocyte proliferation, since it induces a state of quiescence that approaches that of normal human epidermis.

Cell Cycle↗

PubMed2002

Early progression from dimethyl sulfoxide-induced G(0)/G(1) arrest in L(1210) cells.

Recently, dimethyl sulfoxide (DMSO) has been used as a convenient cryoprotectant for stem cells in stem cell transplantation using allogenic peripheral blood or umbilical cord blood. As the stem cells have a multipotency, clarification of the extent of cell proliferation after transplantation is difficult. In the present study, DMSO gradually induced G(0)/G(1) arrest in mouse leukemia L(1210) cells with good cell viability. After removal of DMSO, the cells proliferated appropriately, resulting in expression of the DNA-synthesizing enzymes thymidylate synthase and thymidine kinase within 6h, and the cells entering into S phase within 12h. The sequence was followed by the marked activation of both enzymes within 24h and the increase of bromodeoxyuridine (BrdU) immunoreactive (S phase) cells with rapid cell proliferation within 36 h. In conclusion, mouse leukemia L(1210) cells, which were treated with 1.5% DMSO for 96 h, tolerated the treatment and reversed the cell cycle arrest within 36 h.

Animals↗