Differing responses of G2-related genes during differentiation of HL60 cells induced by TPA or DMSO

Differentiation leads to the cessation of cellular proliferation, but little is known about the molecular mechanisms of growth arrest. We compared the effect of two differentiation inducers, 12-o-tetradecanoyl 13-acetate (TPA) and dimethyl sulfoxide (DMSO) on both the cell-cycle and the modulation of G2-related genes in synchronized HL60 cells. TPA treatment of HL60 cells resulted in G1 arrest within 24 h. In contrast, the cell cycling of DMSO-treated cells was initially accelerated and they progressed to the second cycle before accumulating in the G1 phase. Expression of cyclin B, cdc25, wee1 and cdc2 was studied during cell cycle arrest by Northern blot hybridization. Expression of cyclin B, cdc25 and cdc2 fluctuated in association with cell cycle progression towards the G2/M phase, while wee1 expression remained constant in untreated cells. These four genes were highly expressed in TPA-treated cells for the first 12 h, but drastic down-regulation was seen at 18 h and expression became undetectable after 24 h. In contrast, no remarked changes of gene expression were seen in DMSO-treated cells. These findings suggest that cell cycle progression along with the initial process of differentiation in response to TPA differs from the response to DMSO and that the down-regulation of cdc2 expression by TPA-treated HL60 cells contributes to endorsement of G1 arrest.     

SOCS1 counteracts ROS-mediated survival signals and promotes apoptosis by modulating cell cycle to increase radiosensitivity of colorectal cancer cells

As negative regulators of cytokine signaling pathways, suppressors of cytokine signaling (SOCS) proteins have been reported to possess both pro-tumor and anti-tumor functions. Our recent studies have demonstrated suppressive effects of SOCS1 on epithelial to mesenchymal signaling in colorectal cancer cells in response to fractionated ionizing radiation or oxidative stress. The objective of the present study was to determine the radiosensitizing action of SOCS1 as an anti-tumor mechanism in color-ectal cancer cell model. In HCT116 cells exposed to ionizing radiation, SOCS1 over-expression shifted cell cycle arrest from G2/M to G1 and promoted radiation-induced apoptosis in a p53-dependent manner with down-regulation of cyclin B and up-regulation of p21. On the other hand, SOCS1 knock-down resulted in a reduced apoptosis with a decrease in G1 arrest. The regulatory action of SOCS1 on the radiation response was mediated by inhibition of radiation-induced Jak3/STAT3 and Erk activities, thereby blocking G1 to S transition. Radiation-induced early ROS signal was responsible for the activation of Jak3/Erk/STAT3 that led to cell survival response. Our data col-lectively indicate that SOCS1 can promote radiosensitivity of colorectal cancer cells by counteracting ROS-mediated survival signal, thereby blocking cell cycle progression from G1 to S. The resulting increase in G1 arrest with p53 activation then contributes to the promotion of apoptotic response upon radiation. Thus, induction of SOCS1 expression may increase therapeutic efficacy of radiation in tumors with low SOCS1 levels.     

Effects of ionizing radiation on cyclin expression in HeLa cells.

The levels of cyclin B mRNA and protein rise rapidly in G2 + M phase, and fall at the end of mitosis. The studies described here were initiated to determine the effects of ionizing radiation on the level of cyclin B bearing in mind that the division delay induced by ionizing radiation might be influenced by the expression of cyclin B. After irradiation in S phase, the cyclin B mRNA in HeLa cells was measured as the cells proceeded through the cell cycle. Instead of the usual rise, after irradiation cyclin B mRNA levels remained low during the G2 delay. After irradiation in G2 phase, cyclin B mRNA was readily detectable although at slightly lower levels than in the controls. However, cyclin B protein was markedly decreased in amount.     

Low-dose radiation stimulates the proliferation of normal human lung fibroblasts via a transient activation of Raf and Akt

The biological effects of low-dose radiation have been investigated and debated for more than a century, but its cellular effects and regulatory mechanisms remain poorly understood. This study shows the human cellular responses to low-dose radiation in CCD-18 Lu cells, which are derived from normal human lung fibroblasts. We examined a colony-forming assay for cell survival by ionizing radiation. Live cell counting and cell cycle analysis were measured for cell proliferation and cell cycle progression following low-dose irradiation. We examined Raf and Akt phosphorylation to determine the proliferation mechanism resulting from low-dose radiation. We also observed that p53 and p21 were related to cell cycle response. We found that 0.05 Gy of ionizing radiation enhanced cell proliferation and did not change the progression of the cell cycle. In addition, 0.05 Gy of ionizing radiation transiently activated Raf and Akt, but did not change phospho-p53, p53 and p21 in CCD-18 Lu cells. However, 2 Gy of ionizing radiation induced cell cycle arrest, phosphorylation of p53, and expression of p53 and p21. The phosphorylation of Raf and Akt proteins induced by 0.05 Gy of ionizing radiation was abolished by pre-treatment with an EGFR inhibitor, AG1478, or a PI3k inhibitor, LY294002. Cell proliferation stimulated by 0.05 Gy of ionizing radiation was blocked by the suppression of Raf and Akt phosphorylation with these inhibitors. These results suggest that 0.05 Gy of ionizing radiation stimulates cell proliferation through the transient activation of Raf and Akt in CCD-18 Lu cells.     

TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence.     

Activation-induced T-cell death is cell cycle dependent and regulated by cyclin B.

Developing thymocytes and some T-cell hybridomas undergo activation-dependent programmed cell death. Although recent studies have identified some critical regulators in programmed cell death, the role of cell cycle regulation in activation-induced cell death in T cells has not been addressed. We demonstrate that synchronized T-cell hybridomas, irrespective of the point in the cell cycle at which they are activated, stop cycling shortly after they reach G2/M. These cells exhibit the diagnostic characteristics of apoptotic cell death. Although p34cdc2 levels are not perturbed after activation of synchronously cycling T cells, cyclin B- and p34cdc2-associated histone H1 kinase activity is persistently elevated. This activation-dependent induction of H1 kinase activity in T cells is associated with a decrease in the phosphotyrosine content of p34cdc2. We also demonstrate that transient inappropriate coexpression of cyclin B with p34cdc2 induces DNA fragmentation in a heterologous cell type. Finally, in T cells, cyclin B-specific antisense oligonucleotides suppress activation-induced cell death but not cell death induced by exposure to dexamethasone. We therefore conclude that a persistent elevation of the level of cyclin B kinase is required for activation-induced programmed T-cell death.     

Radiation-induced epigenetic DNA methylation modification of radiation-response pathways

DNA methylation can regulate gene expression and has been shown to modulate cancer cell biology and chemotherapy resistance. Therapeutic radiation results in a biological response to counter the subsequent DNA damage and genomic stress in order to avoid cell death. In this study, we analyzed DNA methylation changes at >450,000 loci to determine a potential epigenetic response to ionizing radiation in MDA-MB-231 cells. Cells were irradiated at 2 and 6 Gy and analyzed at 7 time points from 1–72 h. Significantly differentially methylated genes were enriched in gene ontology categories relating to cell cycle, DNA repair, and apoptosis pathways. The degree of differential methylation of these pathways varied with radiation dose and time post-irradiation in a manner consistent with classical biological responses to radiation. A cell cycle arrest was observed 24 h post-irradiation and DNA damage, as measured by γH2AX, resolved at 24 h. In addition, cells showed low levels of apoptosis 2–48 h post-6 Gy and cellular senescence became significant at 72 h post-irradiation. These DNA methylation changes suggest an epigenetic role in the cellular response to radiation.     

p53-independent downregulation of histone gene expression in human cell lines by high- and low-let radiation

Using microarrays to analyze differential gene expression as a function of p53 status and radiation quality, we observed downregulation of a large set of histone genes in p53 wild-type TK6 cells 24 h after exposure to equitoxic doses of high-LET (1.67 Gy 1 GeV/amu (56)Fe ions) or low-LET (2.5 Gy γ rays) radiation. Quantitative real-time PCR of specific subtypes of core (H2A, H2B, H3 and H4) and linker (H1) histones confirmed this result. DNA synthesis and histone gene expression are tightly coordinated during the S phase of the cell cycle, and both processes are regulated by cell cycle checkpoints in response to DNA damage caused by ionizing radiation. However, we observed similar repression of histone gene expression in both TK6 cells and their p53-null derivative NH32 after radiation exposure, although the histone gene expression was not decreased to the same extent in NH32 cells as it was in TK6 cells. We also found decreased histone gene expression that was dose- and time-dependent in the colon cancer cell line HCT116 and its p53-null derivative. These results show that both high- and low-LET radiation exposure negatively regulate histone gene expression in human lymphoblastoid and colon cancer cell lines independent of p53 status.     

Ectopic Expression of cdc2/cdc28 Kinase Subunit Homo sapiens 1 Uncouples Cyclin B Metabolism from the Mitotic Spindle Cell Cycle Checkpoint

Primary human fibroblasts arrest growth in response to the inhibition of mitosis by mitotic spindle-depolymerizing drugs. We show that the mechanism of mitotic arrest is transient and implicates a decrease in the expression of cdc2/cdc28 kinase subunit Homo sapiens 1 (CKsHs1) and a delay in the metabolism of cyclin B. Primary human fibroblasts infected with a retroviral vector that drives the expression of a mutant p53 protein failed to downregulate CKsHs1 expression, degraded cyclin B despite the absence of chromosomal segregation, and underwent DNA endoreduplication. In addition, ectopic expression of CKsHs1 interfered with the control of cyclin B metabolism by the mitotic spindle cell cycle checkpoint and resulted in a higher tendency to undergo DNA endoreduplication. These results demonstrate that an altered regulation of CKsHs1 and cyclin B in cells that carry mutant p53 undermines the mitotic spindle cell cycle checkpoint and facilitates the development of aneuploidy. These data may contribute to the understanding of the origin of heteroploidy in mutant p53 cells.     

CyclinB1 expression is elevated and mitosis is delayed in HeLa cells expressing autonomous CaMKII

Calcium is a second messenger that is implicated in the regulation of cell cycle transitions. Calmodulin is a ubiquitous protein that translates intracellular calcium signals and activates several enzymes including calcium/calmodulin-dependent protein kinase II (CaMKII). Pharmacological inhibitors and constitutively active mutants have implicated CaMKII in cell cycle mediation. Specifically, constitutively active CaMKII impedes mitosis. In order to elucidate the molecular mechanisms underlying this phenomenon, the effect of constitutively active CaMKII gene expression on cdc2/cyclin B1 was investigated. As seen in previous studies with S. pombe, constitutively active CaMKII-hindered mitosis. However, this report shows that CaMKII does not cause permanent cell cycle arrest but delays progression into mitosis. Constitutive CaMKII expression also leads to elevations in cyclin B1 expression and cdc2 tyrosine-15 phosphorylation, analogous to observations in cells treated with hydroxyurea. Taken together, these data suggest that constitutive CaMKII may delay mitosis by activating a cell cycle checkpoint.     

c-Fos/activator protein-1 transactivates wee1 kinase at G(1)/S to inhibit premature mitosis in antigen-specific Th1 cells

M-phase promoting factor is a complex of cdc2 and cyclin B that is regulated positively by cdc25 phosphatase and negatively by wee1 kinase. We isolated the wee1 gene promoter and found that it contains one AP-1 binding motif and is directly activated by the immediate early gene product c-Fos at cellular G(1)/S phase. In antigen-specific Th1 cells stimulated by antigen, transactivation of the c-fos and wee1 kinase genes occurred sequentially at G(1)/S, and the substrate of wee1 kinase, cdc2-Tyr15, was subsequently phosphorylated at late G(1)/S. Under prolonged expression of the c-fos gene, however, the amount of wee1 kinase was increased and its target cdc2 molecule was constitutively phosphorylated on its tyrosine residue, where Th1 cells went into aberrant mitosis. Thus, an immediate early gene product, c-Fos/AP-1, directly transactivates the wee1 kinase gene at G(1)/S. The transient increase in c-fos and wee1 kinase genes is likely to be responsible for preventing premature mitosis while the cells remain in the G(1)/S phase of the cell cycle.     

PCNA等5种基因在小鼠睾丸发育过程中的表达

以出生1日、30日和成年昆明小鼠睾丸组织为实验材料,利用地高辛标记的基因探针在组织切片上进行DNA－mRNA分子原位杂交,研究了PCNA、cyclinD1、cdc2、P21和P16基因共5种细胞周期调控基因在小鼠睾丸发育中的表达变化。结果发现：PCNA基因在30日龄和成年鼠睾丸中都有强的表达;而cyclin D1基因只在30日龄的小鼠睾丸组织中有表达;P21、P16和cdc2基因在3个不同的发育阶段都没有表达。这些结果表明：(1)细胞周期调控基因cyclin D1、cdc2、P16和P21与小鼠睾丸发育和精子发生过程的细胞增殖控制关系不大,可能小鼠睾丸和精子发育过程中的细胞增殖调控与其他细胞的增殖调控有不同的机制;(2)cyclin D1基因在小鼠睾丸中的表达模式表明,cyclin D1基因在睾丸发育中有不同于细胞增殖促进的作用;(3)小鼠睾丸发育中,精子发生开始时期可能晚于30日龄。 Abstracts：Using in situ hybridization technique with Dig Labeled DNA probe, we studied the expression of PCNA,cyclin D1, cdc2, P21 and P16 gene in the test of one day old,30 days old and adult Kunming mouse.The results shows that the expression of PCNA gene was strong in the test of 30 days old and adult mouse; cyclin D1 gene only expressed in the test of 30 days old mouse; cdc2,P21 and P16 gene did not express in the test of all three groups of mouse.These results suggest that:(1)cyclin D1,cdc2,P16 and P21 genes are not related to the regulaton of cell cycle during test development and germ genaration, and possibly the machanism of cell cycle regulation during test development and germ genaration is different from other kind of cells;(2)cyclin D1 has other founctions except promoting cell profilation;and (3)the germ genaration during test development may start after 30 days old.     

Differential gene expression in gamma-irradiated BALB/3T3 fibroblasts under the influence of 3-aminobenzamide,an inhibitior of parp enzyme

3-Aminobenzamide (3AB) is an inhibitor of poly (ADP-ribose) polymerase (PARP), an enzyme implicated in the maintenance of genomic integrity, which is activated in response to radiation-induced DNA strand breaks. cDNA macroarray membranes containing 1536 clones were used to characterize the gene expression profiles displayed by mouse BALB/3T3 fibroblasts (A31 cell line) in response to ionizing irradiation alone or in combination with 3AB. A31 cells in exponential growth were pre-treated with 3AB 4mM 1h before gamma-irradiation (4Gy), remaining in culture during 6h until harvesting time. A31 cells treated with 3AB alone presented a down-regulation in genes involved in protein processing and cell cycle control, while an up-regulation of genes involved in apoptosis and related to DNA/RNA synthesis and repair was verified. A31 cells irradiated with 4Gy displayed 41 genes differentially expressed, being detected a down-regulation of genes involved in protein processing and apoptosis, and genes controlling the cell cycle. Concomitantly, another set of genes for protein processing and related to DNA/RNA synthesis and repair were found to be up-regulated. A positive or negative interaction effect between 3AB and radiation was verified for 29 known genes. While the combined treatment induced a synergistic effect on the expression of LCK proto-oncogene and several genes related to protein synthesis/processing, a negative interaction effect was found for the expression of genes related to cytoskeleton and extracellular matrix assembly (SATB1 and Anexin III), cell cycle control (tyrosine kinase), and genes participating in DNA/RNA synthesis and repair (RNA helicase, FLAP endonuclease-1, DNA-3 glycosylase methyladenine, splicing factor SC35 and Soh1). The present data open the possibility to investigate the direct participation of specific genes, or gene products acting in concert in the mechanism underlying the cell response to radiation-induced DNA damage under the influence of PARP inhibitor.     

Changes in MicroRNA expression patterns in human fibroblasts after low-LET radiation

Exposure to radiation provokes cellular responses controlled in part by gene expression networks. MicroRNAs (miRNAs) are small non-coding RNAs which mostly regulate gene expression by degrading the messages or inhibiting translation. Here, we investigated changes in miRNA expression patterns after low (0.1 Gy) and high (2.0 Gy) doses of X-ray in human fibroblasts. At early (0.5 h) and late (6 and 24 h) time points, irradiation caused qualitative and quantitative differences in the down-regulation of miRNA levels, including miR-92b, 137, 660, and 656. A transient up-regulation of miRNAs was observed after 2 h post-irradiation following high doses of radiation, including miR-558 and 662. MicroRNA levels were inversely correlated with targets from mRNA and proteomic profiling after 2.0 Gy of radiation. MicroRNAs miR-579, 608, 548-3p, and 585 are noted for targeting genes involved in radioresponsive mechanisms, such as cell cycle checkpoint and apoptosis. We suggest here a model in which miRNAs may act as "hub" regulators of specific cellular responses, immediately down-regulated so as to stimulate DNA repair mechanisms, followed by up-regulation involved in suppressing apoptosis for cell survival. Taken together, miRNAs may mediate signaling pathways in sequential fashion in response to radiation, and may serve as biodosimetric markers of radiation exposure.     

TPR proteins required for anaphase progression mediate ubiquitination of mitotic B-type cyclins in yeast.

The abundance of B-type cyclin-CDK complexes is determined by regulated synthesis and degradation of cyclin subunits. Cyclin proteolysis is required for the final exit from mitosis and for the initiation of a new cell cycle. In extracts from frog or clam eggs, degradation is accompanied by ubiquitination of cyclin. Three genes, CDC16, CDC23, and CSE1 have recently been shown to be required specifically for cyclin B proteolysis in yeast. To test whether these genes are required for cyclin ubiquitination, we prepared extracts from G1-arrested yeast cells capable of conjugating ubiquitin to the B-type cyclin Clb2. The ubiquitination activity was cell cycle regulated, required Clb2's destruction box, and was low if not absent in cdc16, cdc23, cdc27, and cse1 mutants. Furthermore all these mutants were also defective in ubiquitination of another mitotic B-type cyclin, Clb3. The Cdc16, Cdc23, and Cdc27 proteins all contain several copies of the tetratricopeptide repeat and are subunits of a complex that is required for the onset of anaphase. The finding that gene products that are required for ubiquitination of Clb2 and Clb3 are also required for cyclin proteolysis in vivo provides the best evidence so far that cyclin B is degraded via the ubiquitin pathway in living cells. Xenopus homologues of Cdc16 and Cdc27 have meanwhile been shown to be associated with a 20S particle that appears to function as a cell cycle-regulated ubiquitin-protein ligase.     

Cyclin B (p56cdc13) localization in the yeast Schizosaccharomyces pombe: An ultrastructural and immunocytochemical study

Summary— The eucaryote cell cycle is driven by a set of cyclin dependent kinases (CDKs) associated to cyclins, which confer not only the activity but also the substrate specificity and the proper localization of the kinase activity. In the fission yeast Schizosaccharomyces pombe, only one cyclin, the product of the cdc13 gene (p56^cdc13), is required to be associated with p34^cdc2, to control the complete cell cycle. Earlier studies have localized this complex mainly in the nucleus and its periphery. Using new improved electron microscopy (EM) technologies, based on high pressure freezing fixation, we refined previous studies, evidencing cytoplasmic localization of p56^cdc13, in addition to the nuclear localization previously observed. Further immunofluorescence studies, performed on aldehydically fixed cells, confirmed our EM results, emphasizing the major cytoplasmic localization of p56^cdc13 in interphase cells and the relocalization towards the nucleus in mitotic cells, suggesting that the S pombe cyclin B localization is cell cycle-regulated.