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At least 1,045 records · Page 58Linked to original sources
PubMed2006

Induction of p21CIP/WAF-1 and G2 arrest by ionizing irradiation impedes caspase-3-mediated apoptosis in human carcinoma cells.

There is an ongoing controversy regarding the relevance of apoptosis induction by ionizing irradiation as compared with other end points including transient or permanent cell cycle arrest of damaged cells. Here, we show that such permanent cell cycle arrest and apoptosis represent two sides of the same coin. MCF-7 cells fail to express procaspase-3, which results in resistance to apoptosis induced by anticancer drugs. Conversely, restoration of procaspase-3 sensitizes MCF-7 cells to chemotherapeutics including epirubicine, etoposide and taxol. In contrast, irradiation does not trigger apoptotic cell death but results in prolonged arrest in the G2 phase of the cell division cycle regardless of procaspase-3 expression. This suggested that the propensity of MCF-7 cells to arrest at the G2 checkpoint results in resistance to apoptosis upon gamma-irradiation. This G2 arrest was associated with upregulation of p21CIP/WAF-1. Inhibition of DNA-damage-induced stress kinases and p21CIP/WAF-1 expression by caffeine abrogated G2 arrest and induced apoptosis of the irradiated cells in a caspase-3-dependent manner. Inhibition of cell cycle progression by adenoviral expression of the cyclin dependent kinase inhibitor p21CIP/WAF-1 prevented apoptosis upon caffeine treatment indicating that cell cycle progression, that is, G2-release, is required for induction of apoptosis. Likewise, cells homozygously deleted for p21CIP/WAF-1 (HCT116 p21-/-) display enhanced irradiation-induced apoptosis via a caspase-3-dependent mechanism. These data indicate that the disruption of G2 checkpoint control overcomes cell cycle arrest and resistance to gamma-irradiation-induced cell death. Thus, DNA damage may trigger a permanent G2 arrest as an initial inactivation step of tumor cells where the phenomenon of apoptosis is hidden unless cell cycle arrest is overcome. The efficient induction of apoptosis upon G2 release thereby depends on the propensity to activate the key executioner caspase-3. This finding is of crucial importance for the understanding of molecular steps underlying the efficacy of ionizing radiation to delete tumor cells.

Antineoplastic Agents↗
PubMed2001

A cell-cycle-regulated kinase activity phosphorylates plant retinoblastoma protein and contains, in Arabidopsis, a CDKA/cyclin D complex.

The activity of cyclin-dependent kinases (CDK) is crucial for cell-cycle transitions. Here, we report the identification of a CDK activity that phosphorylates the retinoblastoma-related (RBR) protein. A CDK/cyclin complex that binds to and phosphorylates RBR may be isolated from various plant sources, e.g. wheat, maize, Arabidopsis thaliana and tobacco, and from cells growing under various conditions. The presence of an RBR-associated CDK activity correlates with the proliferative activity, suggesting that phosphorylation of RBR is a major event in actively proliferating tissues. In A. thaliana, this activity comprises a PSTAIRE CDKA and at least cyclin D2. Furthermore, this CDK activity is cell-cycle-regulated, as revealed by studies with highly synchronized tobacco BY-2 cells where it is maximal in late G1 and early S phase cells and progressively decreases until G2 phase. Aphidicolin-arrested but not roscovitine-arrested cells contain a PSTAIRE-type CDK that binds to and phosphorylates RBR. Thus, association with a D-type cyclin is a likely mechanism leading to CDK activation late in G1. Our studies constitute the first report measuring the activity of CDK/cyclin complexes formed in vivo on RBR, an activity that fluctuates in a cell-cycle-dependent manner. This work provides the basis for further studies on the impact of phosphorylation of RBR on its function during the cell cycle and development.

CDC2-CDC28 Kinases↗
PubMed1977

Microviscosity modulation during the cell cycle of neuroblastoma cells.

Microviscosity (n) of the cell membrane lipid layer was determined in synchronized C1300 mouse neuroblastoma cells (clone Neuro-2A) by fluorescence polarization of 1,6-diphenystratum. The determined n value was maximal in mitosis, decreased markedly in the G1 phase, remained constant at a low level during the S phase, and increased again during the G2 phase. These findings imply a direct role of the cell membrane fluidity in regulation of the cell cycle.

Animals↗
PubMed1998

Suppression of adenovirus E1A-induced apoptosis by mutated p53 is overcome by coexpression with Id proteins.

The rat 3Y1 derivative cell lines, EId10 and EId23, established by introducing the adenovirus E1A12S, Id-1H, and Id-2H cDNAs linked to the hormone-inducible promoter, express these proteins upon treatment with dexamethasone and elicit apoptosis, although these cell lines express mutated p53. The E1A mutants containing a deletion in either the N terminus or the conserved region 1 were unable to induce apoptosis in cooperation with Ids. Western blot analysis of the immunoprecipitates prepared from the dexamethasone-treated EId10 cell extract showed that Id-2H preferentially binds to E1A and E2A (E12/E47) helix-loop-helix transcription factors in vivo, but scarcely to the retinoblastoma protein. After induction of E1A and Ids, EId10 and EId23 cells began to accumulate in S phase and undergo apoptosis before entering G2 phase, suggesting that abnormal synthesis of DNA induced by coexpression of E1A, Id-1H, and Id-2H results in the induction of apoptosis. Apoptosis also is induced in mouse A40 (p53-/-) cells by E1A alone or E1A plus Ids after transient transfection of the expression vectors. The induction of apoptosis is stimulated by coexpression with wild-type p53; however, apoptosis induced by E1A alone was suppressed completely by coexpression with mutated p53, whereas apoptosis induced by E1A plus Ids was stimulated by the mutated p53 as done by wild-type p53. These results suggest that the suppressive function of mutated p53 is overcome by Ids.

Adenovirus E1A Proteins↗
PubMed2002

Activation of the ribosomal RNA genes late in the third cell cycle of porcine embryos.

In porcine embryos, nucleoli are first observed during the third postfertilization cell cycle, i.e., at the 4-cell stage. However, direct studies of the initiation of rRNA transcription have not been reported. This transcription was investigated in the present study by simultaneous visualization of the rRNA genes and the rRNA by fluorescent in situ hybridization using a porcine 28S rDNA probe and subsequent visualization of argyrophilic nucleolar proteins by silver staining of extracted and fixed nuclei from in vivo-derived porcine embryos (n = 229). Nucleologenesis was observed by transmission electron microscopy. In general, the 2-cell and 4-cell embryos fixed at 10 and 20 h postcleavage (hpc) showed no signs of rRNA transcription. Four small clusters of fluorescein isothiocyanate (FITC) labeling were visible in interphase nuclei, consistent with hybridization to the rRNA gene clusters only; there was no silver staining at the sites of the rRNA genes and nucleolus precursor bodies. From 30 hpc onwards, most 4-cell embryos had medium size to large clusters of FITC-labeled areas colocalized with silver staining of rRNA gene clusters and fibrillogranular nucleoli. These observations indicate that rRNA transcription had been initiated. These signs of rRNA synthesis could be blocked by actinomycin D, which is a strong inhibitor of RNA polymerase I. The rRNA transcription of porcine embryos is initiated between 20 and 30 hpc, corresponding to the end of the S-phase or the beginning of the G2 phase during the third cell cycle.

Animals↗
PubMed1997

ORC-dependent and origin-specific initiation of DNA replication at defined foci in isolated yeast nuclei.

We describe an in vitro replication assay from yeast in which the addition of intact nuclei to an S-phase nuclear extract results in the incorporation of deoxynucleotides into genomic DNA at spatially discrete foci. When BrdUTP is substituted for dTTP, part of the newly synthesized DNA shifts to a density on CsCl gradients, indicative of semiconservative replication. Initiation occurs in an origin-specific manner and can be detected in G1- or S-phase nuclei, but not in G2-phase or mitotic nuclei. The S-phase extract contains a heat- and 6-DMAP-sensitive component necessary to promote replication in G1-phase nuclei. Replication of nuclear DNA is blocked at the restrictive temperature in an orc2-1 mutant, and the inactive Orc2p cannot be complemented in trans by an extract containing wild-type ORC. The initiation of DNA replication in cln-deficient nuclei blocked in G1 indicates that the ORC-dependent prereplication complex is formed before Start. This represents the first nonviral and nonembryonic replication system in which DNA replication initiates in an ORC-dependent and origin-specific manner in vitro.

Cell Cycle↗
PubMed1977

Commitment to deoxyribonulceic acid synthesis and the cell cycle in endotoxin-stimulated murine spleen cells.

Events associated with endotoxin-induced mitogenesis in murine spleen cells were investigated. Commitment to deoxyribonucleic acid (DNA) synthesis, the onset of DNA synthesis, and phases of cell cycle were timed. Increased levels of DNA synthesis in murine spleen cells stimulated with endotoxin were observed 12 to 16 h after the addition of the mitogen. The total cell cycle time of stimulated B-cells was 11 to 14 h. The S-phase was 8 h. The G2-phase was 1 h, and the combined M-plus G1-phase of cycling cells was 2 to 5 h. A 1- to 4-h exposure to lipopolysaccharide elicited a significant increase in DNA synthesis. Progressively longer exposures to lipopolysaccharide, up to 24 h, produced further increases in first-cycle DNA synthesis. Polymyxin B, when added with endotoxin to cultures from the outset, inhibited first-cycle DNA synthesis. However, if addition of the antibiotic was delayed, progressive increases in first-cycle dna synthesis were observed. These data indicate a heterogeneity among B-cells in their responsiveness to endotoxin.

Animals↗
PubMed1996

Drosophila NAP-1 is a core histone chaperone that functions in ATP-facilitated assembly of regularly spaced nucleosomal arrays.

We describe the cloning and analysis of Drosophila nucleosome assembly protein 1 (dNAP-1), a core histone-binding protein that functions with other chromatin assembly activities in a Drosophila chromatin assembly factor 1-containing fraction (dCAF-1 fraction) in the ATP-facilitated assembly of regularly spaced nucleosomal arrays from purified core histones and DNA. Purified, recombinant dNAP-1 acts cooperatively with a factor(s) in the dCAF-1 fraction in the efficient and DNA replication-independent assembly of chromatin. In the presence of histone H1, the repeat length of the chromatin is similar to that of native chromatin from Drosophila embryos. By coimmunoprecipitation analysis, dNAP-1 was found to be associated with histones H2A and H2B in a crude whole-embryo extract, which suggests that dNAP-1 is bound to the histones in vivo. Studies of the localization of dNAP-1 in the Drosophila embryo revealed that the factor is present in the nucleus during S phase and is predominantly cytoplasmic during G2 phase. These data suggest that NAP-1 acts as a core histone shuttle which delivers the histones from the cytoplasm to the chromatin assembly machinery in the nucleus. Thus, NAP-1 appears to be one component of a multifactor chromatin assembly machinery that mediates the ATP-facilitated assembly of regularly spaced nucleosomal arrays.

Adenosine Triphosphate↗
PubMed1989

Double labeling with iodo- and bromodeoxyuridine for cell kinetics studies.

The rate of progression through the cell cycle was determined in five human glioma cell lines by a new sequential immunohistochemical staining technique. The cells were labeled first with iododeoxyuridine (IdUrd) for 1-3 hr and then with bromodeoxyuridine (BrdUrd) for 30 min. Labeled cells were identified with Br-3, a monoclonal antibody that recognizes only BrdUrd, and with IU-4, an antibody that recognizes both IdUrd and BrdUrd. Each slide was stained sequentially, first with the immunoperoxidase method for Br-3 and then with the alkaline phosphatase-anti-alkaline phosphatase method for IU-4. Cells that were positive only for IU-4 represented the fraction of S-phase cells that passed into the G2 phase during the period of incubation with IdUrd. The rates of progression measured by this method were constant in each cell line and resulted in smaller standard errors than were obtained by measurements from specimens stained singly for IdUrd and BrdUrd in different slides. The duration of the S-phase calculated from this fraction in the five cell lines ranged from 8-13 hr; the estimated potential doubling times were 25-32 hr and were very similar to the actual doubling times.

Bromodeoxyuridine↗
PubMed1980

More evidence for replication-transcription-coupling in Physarum polycephalum.

Endogenous RNA polymerase activity of isolated nuclei from Physarum polycephalum was determined at high (400 mM KCl) and low (5--100 mM KCl) ionic strength. The activity of RNA polymerase B (alpha-amanitin-sensitive UMP incorporation) and of RNA polymerase A (plus C) (alpha-amanitin-resistant UMP incorporation) was compared in accurately sized nuclear samples derived from macroplasmodia at distinct points of the mitotic cycle. Minimum total RNA polymerase activity was detected in metaphase nuclei. A constant level of RNA polymerase B activity was detected at all other stages of the mitotic cycle, if nuclei were assayed at high ionic strength. However, a high level in S-phase, a low level in G2-phase and again a high level in early prophase were measured, if nuclei were assayed at low ionic strength. Inhibition of DNA synthesis by hydroxyurea in vivo had a selective and drastic effect on in vitro RNA polymerase activity of isolated nuclei derived from S-phase plasmodia, yielding up to 100% inhibition in early S-phase.

Cell Cycle↗
PubMed2004

Analysis of checkpoint responses to histone deacetylase inhibitors.

Cell cycle checkpoints respond to a wide range of stresses to prevent compromise to the integrity of the cell. The best studied checkpoints are those induced by genotoxic agents that cause DNA damage. Histone deacetylase inhibitors not only increase the acetylation state of chromatin histones, but they also perturb the cell cycle, causing both G1 and G2 phase arrests, the latter by initiating a checkpoint response. In this chapter we will describe the analysis of the histone deacetylase inhibitor-sensitive G2 checkpoint using synchronized cell populations.

Bromodeoxyuridine↗
PubMed2006

Thiazolidinediones ameliorate diabetic nephropathy via cell cycle-dependent mechanisms.

Thiazolidinediones are ligands for peroxisome proliferator-activated receptor (PPAR)-gamma, widely used as insulin sensitizer in type 2 diabetic patients and implicated in apoptosis, cell proliferation, and cell cycle regulation. Here, the effect of thiazolidinediones on G1-phase cell cycle arrest, the hallmark in diabetic nephropathy, was investigated. Eight-week-old male Otsuka Long-Evans Tokushima fatty rats were treated with pioglitazone (1 mg x kg body wt(-1) x day(-1)) until 50 weeks of age and compared with insulin treatment. Although similar HbA(1c) levels were observed in both groups, pioglitazone significantly inhibited glomerular hypertrophy and mesangial matrix expansion and reduced urinary albumin excretion compared with the insulin-treated group. In addition, pioglitazone significantly reduced the number of glomerular p27(Kip1)-positive cells. Because prominent expression of PPAR-gamma was observed in podocytes in glomeruli and cultured cells, conditionally immortalized mouse podocyte cells were cultured under 5.5 and 25 mmol/l D-glucose supplemented with pioglitazone. Pioglitazone inhibited cell hypertrophy revealed by [(3)H]thymidine and [(3)H]proline incorporation, and pioglitazone reversed high glucose-induced G1-phase cell cycle arrest, i.e., an increase in G0/G1 phase and decrease in S and G2 phases. Pioglitazone suppressed high glucose-induced phosphorylation of p44/42 mitogen-activated protein kinase and reduced Bcl-2 and p27(Kip1) protein levels. Besides glucose-lowering action, pioglitazone ameliorates diabetic nephropathy via cell cycle-dependent mechanisms.

Albuminuria↗
PubMed1998

Effects of spin labeled derivatives of podophyllotoxin on cell cycle and macromolecular synthesis in human lymphoid leukemia Molt 4B cells.

AIM: To examine the effect of the spin labeled derivatives of podophyllotoxin, N-podophyllic acid-N"-[4-(2,2,6,6-tetramethyl-1-piperidinyloxy)] thiosemicarbazide (GP4) and 4-[4"-(2",2",6",6"-tetramethyl-1"-piperidinyloxy) amino]-4'-demethylepipodophyllotoxin (GP7) on the cell cycle and macromolecular synthesis of human lymphoid leukemia Molt 4B cells in vitro. METHODS: MTT assay, 3H incorporation, and flow cytometer were used. RESULTS: GP4, GP7, and etoposide 0.02-100 mmol.L-1 cultured for 48 h inhibited the proliferation of human lymphoid leukemia Molt 4B cells. IC50 values of GP4, GP7, and etoposide were 0.11, 4.7, and 1.6 mmol.L-1, respectively. DNA and protein syntheses were obviously suppressed by GP4, GP7, and etoposide 10 mmol.L-1 for 48 h. After Molt 4B cells were treated with GP4, GP7, and etoposide 10 mmol.L-1 for 6 and 12 h, the mitotic index was increased by GP4 and reduced by GP7 and etoposide. According to flow cytometric BrdU/DNA analysis, GP4 slightly retarded S phase and mainly arrested cell cycle progression in G2/M phase, whereas GP7 similar to etoposide induced cells accumulated at S phase and retarded the cells in G2 phase. CONCLUSION: GP4 and GP7 inhibit the proliferation of Molt 4B cells, but the mechanisms are different.

Antineoplastic Agents, Phytogenic↗
PubMed1999

[Expression and regulation of p16 in osteosarcoma].

OBJECTIVE: To study the expression and regulation of p16 and related factors in osteosarcoma. METHODS: The levels of p16 protein and related factors were explored by Western blot analysis in 12 cases of osteosarcoma tissues and in cells of different phases of osteosarcoma cell line OS732. The status of p16 protein in osteosarcoma cells was revealed by immunofluorescence analysis. RESULTS: p16 protein was detected in 10 of 12 osteosarcoma tissues. pRb was found in all of the specimens, but 8 of them were hyperphosphorylated. p16 protein was the least in G1 phase. Localized in cytoplasm during G1 phase, p16 protein gathered around nuclear envelope in S phase and entered the nucleus in G2 phase. CONCLUSIONS: The expression rate of p16 protein was 83.3% in osteosarcoma tissues. The expression of p16 protein was unable to inhibit the phosphorylation of pRb in most osteosarcomas; p16 protein was extremely low in G1 phase, presumably it was sufficient to inhibit the phosphorylation of pRb, causing abnormal proliferation of the cells.

Adolescent↗
PubMed1981

Cell cycle-specific inhibition by retinoic acid of xenotropic murine retrovirus expression.

Several retinoids were examined for their capacity to block chemically induced expression of endogenous xenotropic retrovirus from Kirsten sarcoma virus-transformed BALB/c mouse cells. Retinoic acid (RA) was found to inhibit induction of virus by 5-iododeoxyuridine, cycloheximide, and histidinol; inhibition was concentration (10(-4) to 10(-6) M) and time dependent (1 to 7 hr) and not a consequence of cytotoxicity. Following a 6-hr treatment with 10(-4) M RA, [3H]thymidine and [3H]uridine incorporation into total cellular DNA and RNA was reduced 37 and 63%, respectively. Heteronuclear RNA synthesis was reduced 36 and 7% within 4 hr by 10(-4) and 10(-5) M RA, respectively, indicating that inhibition was not the result of a general transcriptional block. Using synchronized cells, it was found that 5 X 10(-5) M RA added in G1 phase and followed by cycloheximide or 5-iododeoxyuridine induction inhibited virus expression 60 and 84%, respectively. Little or no inhibition was observed when RA was added during S phase with the inducers or during G2 phase followed by inducers. Cells synchronized by mitotic arrest showed a RA-mediated restriction point in early-to-mid-G1 phase as indicated by a delay in the onset of DNA synthesis and an inhibition of virus induction during S phase. The results show the presence in Kirsten sarcoma virus-transformed BALB/c cells of a RA-sensitive G1 restriction point for cell progression and suggest that inhibition of retrovirus activation may be related to an extended G1 phase.

Animals↗
PubMed1995

A variant form of cyclin-dependent kinase 2 (Cdk2) in a malignantly transformed rat thyroid (FRTL-Tc) cell line.

Cyclin-dependent kinase 2 (Cdk2) controls the transition from the G1 to the S phase in the mammalian cell cycle. We found by immunoblotting that anti-Cdk2 antibodies recognize three Cdk2 proteins (of 33, 34 and 39 kDa) in FRTL-5 and FRTL-Tc cells (malignantly transformed FRTL cells). Although 33 kDa protein is a phosphorylated form of 34 kDa protein previously reported, the nature of 39 kDa protein is unknown. In order to determine the nature of this protein, we screened a FRTL-5 cDNA library. Two cDNA clones of the rat homologue (rat Cdk2-alpha and -beta) of human Cdk2 were isolated. The open reading frame of rat Cdk2-alpha cDNA encoded a protein with 428 amino acids and has a high degree of conservation with human Cdk2. The calculated molecular weight of Cdk2-alpha protein is 33892 Da. The rat Cdk2-beta cDNA was identical to Cdk2-alpha cDNA except that it had extra 144 bp; this coincided with insertion of 48 amino acids into Cdk2-alpha protein between Met 196 and Val 197. The calculated molecular weight of Cdk2-beta protein is 39087 Da. Northern blot analysis indicated that the sizes of rat Cdk2-alpha and -beta mRNAs are approximately 2.5 kb and 3.0 kb, respectively. Partial proteolytic mapping showed that Cdk2-beta gene product is 39 kDa Cdk2 in the immunoblotting. We also found that Cdk2-beta protein binds to cyclin A and suc1 proteins. During G1-S phase in FRTL-Tc cells, Cdk2-alpha protein level is constant, but is gradually phosphorylated. In contrast, the level of Cdk2-beta protein increases through the S phase and decreases at the early G2 phase. These results suggest that a variant form of Cdk2 protein might be required for entry into the S phase of the cell cycle in FRTL-Tc cells.

Amino Acid Sequence↗
PubMed1993

Interaction between replication forks and topoisomerase I-DNA cleavable complexes: studies in a cell-free SV40 DNA replication system.

The extreme S-phase-specific cytotoxicity of camptothecin has been shown to involve active DNA replication. To investigate the role of DNA replication in camptothecin cytotoxicity, we have studied the interaction between the DNA replication machinery and the topoisomerase I-camptothecin-DNA ternary cleavable complex in a cell-free SV40 DNA replication system. The formation of topoisomerase I-camptothecin-DNA-cleavable complexes on the replication template efficiently and irreversibly inhibited DNA replication. Two aberrant forms of replication products were produced whose abundance varied with the concentrations of exogenously added topoisomerase I and camptothecin. At low concentrations of topoisomerase I and camptothecin, the major aberrant DNA replication product was close-to-unit-length-linear DNA, while at higher concentrations the predominant product was close-to-dimer-size-linear DNA. Analysis of these aberrant replication products has suggested a "collision" model in which the interaction between an advancing replication fork and a topoisomerase I-camptothecin-DNA-cleavable complex results in irreversible arrest of the replication fork and the formation of a double-strand DNA break at the fork. Concomitant with fork arrest and fork breakage, the reversible cleavable complex was converted into a topoisomerase I-linked DNA break. We propose that one or several of these events triggers S-phase-specific cell killing and G2-phase cell cycle arrest.

Camptothecin↗
PubMed1996

Fanconi anemia genes act to suppress a cross-linker-inducible p53-independent apoptosis pathway in lymphoblastoid cell lines.

Hypersensitivity to cross-linking agents such as mitomycin C (MMC) is characteristic of cells from patients suffering from the inherited bone marrow failure syndrome. Fanconi anemia (FA). Here, we link MMC hypersensitivity of Epstein-Barr virus (EBV)-immortalized FA lymphoblasts to a high susceptibility for apoptosis and p53 activation. In MMC-treated FA cells belonging to complementation group C (FA-C), apoptosis followed cell cycle arrest in the G2 phase. In stably transfected FA-C cells, plasmid-driven expression of the wild-type cytoplasmic FAC protein relieved MMC-dependent G2 arrest and suppressed p53 activation. However, in both FA and non-FA lymphoblasts, p53 seemed not to be instrumental in the induction of MMC-dependent apoptosis, since overexpression of a dominant-negative p53 mutant failed to affect cell survival. In addition, no differences in the level of Bcl-2 expression, an inhibitor of apoptosis, were detected between FA and non-FA cells either in the absence or presence of MMC. Our findings suggest that FAC and the other putative FA gene products may function in a yet to be identified p53-independent apoptosis pathway.

Apoptosis↗