Arrest of the cell cycle reduces susceptibility of target cells to perforin-mediated lysis

Cytotoxic T lymphocytes secrete a pore-forming cytolysin, perforin, that damages membranes of target cells. They also ligate Fas receptors on target cells and provoke apoptotic death. A20 (B lymphoma) and P815 (mastocytoma) cell lines were examined for their susceptibility to perforin-mediated lysis and to Fas-induced apoptosis after blockade of the cell cycle at the G₁/S interface. Cells were arrested at the G₁/S interface by inhibition of DNA synthesis with thymidine or aphidicolin. Subsequently, the treated cells were incubated either with CTL cytotoxic granules or the Fas-specific monoclonal antibody Jo-2. We show that arrest of the cell cycle at the G₁/S interface markedly reduced the susceptibility of target cells to perforin-mediated lysis. In contrast, growth arrest with thymidine or aphidicolin increased susceptibility of A20 and P815 cells to Fas-mediated apoptosis. Susceptibility to lysis by intact CTLs was not affected significantly by blockade of target cells with aphidicolin or thymidine. When cells surviving exposure to perforin-containing granules were isolated on Ficoll density gradients and cell-cycle profiles were examined by flow cytometry, the ratio of G₁ to G₂cells increased among the survivors exposed to granules in contrast to controls incubated with buffer alone. The data suggest that cells in G₁ phase of the cell cycle are less susceptible to the perforin pathway than cells in G₂and S phases but are more susceptible to the Fas pathway. J. Cell. Biochem. 69:425–435, 1998. © 1998 Wiley-Liss, Inc.     

Genetic evidence that aphidicolin inhibits in vivo DNA synthesis in Chinese hamster ovary cells

Using a genetic approach, Chinese hamster ovary (CHO) cells sensitive (aph^S) and resistant (aph^R) to aphidicolin were grown in the presence or absence of various DNA polymerase inhibitors, and the newly synthesized DNA isolated from [³²P]dNMP-labelled, detergent-permeabilized cells, was characterized after fractionation by gel electrophoresis. The particular aph ^Rmutant CHO cell line used was one selected for resistance to aphidicolin and found to possess an altered DNA polymerase of the a-family. The synthesis of a 24 kb replication intermediate was inhibited in wild-type CHO cells grown in the presence of aphidicolin, whereas the synthesis of this replication intermediate was not inhibited by this drug in the mutant CHO cells or in the aphidicolin-resistant somatic cell hybrid progeny constructed by fusion of wild-type and mutant cell lines. Arabinofuranosylcytosine (ara-C), like aphidicolin, inhibited the synthesis of this 24 kb DNA replication intermediate in the wild-type CHO cells but not in the aph^R mutant cells. However, carbonyldiphosphonate (COMDP) inhibited the synthesis of the 24 kb replication intermediate in both wild-type and mutant cells. N²-(p-n-Butylphenyl)-2 deoxyguanisine-5-triphosphate (BuPdGTP) was found to inhibit the formation of Okazaki fragments equally well in the wild-type and mutant cell lines and thus led to inhibition of synthesis of DNA intermediates in both cases. It appears that aphidicolin and ara-C both affect a common target on the DNA polymerase, which is different from that affected by COMDP in vivo. These data also show that aphidicolin, ara-C and COMDP affect the elongation activity of DNA polymerase but not the initiation activity of the enzyme during DNA replication. This is the first report of such differentiation of the DNA polymerase activities during nuclear DNA replication in mammalian cells. The method of analysis described here for replication intermediates can be used to examine the inhibitory activities of other chemicals on DNA synthesis.     

Analysis of DNA size,content and cell cycle in leaves of Napier grass (Pennisetum purpureum Schum.)

Summary Mesophyll cell nuclei isolated from leaves of Pennisetum purpureum were analysed by flow cytometry to determine the nuclear DNA content and the percentage of cells in different phases of the cell cycle. Samples taken from base, middle and tip regions of leaves 2 to 8 (leaf 1, which was adjacent to the meristem, was too small to sample) showed no significant differences in the amount of DNA per G₁ nucleus due to either age or position. The average amount of DNA per G₁ nucleus was 5.78 pg. Although the majority of cells for each sample were in G₁, samples taken from older leaves had higher percentages of cells in G₂ and S phases. More specifically, base and middle regions of older leaves had a higher percentage of cells in G₂ than all three positions in younger leaves. Electrophoretic analysis of nuclear DNA from leaves 2 to 7 showed no evidence of degradation or difference in fragment size for any sample or position. This study was compared to previous work on the relationship between leaf age and embryogenic competence in Pennisetum purpureum. The results suggest that changes in the cell cycle, and/or a loss or fragmentation of the nuclear DNA, are not responsible for loss of embryogenic competence in mature leaf tissue.     

Cessation of Nuclear DNA Synthesis in Differentiating Naegleria*

SYNOPSIS. DNA synthesis during growth and differentiation in Naegleria gruberi strain NEG populations has been studied. Autoradiography of cells labeled with [³H]thymidine revealed that grains are concentrated over the nuclei in logarithmically growing populations of cells, whereas in differentiating cells, grains are scattered over the cytoplasm; i.e. no significant nuclear labeling is detectable. It was established by MAK chromatographic analysis that [³H]thymidine is incorporated into double-stranded DNA in Naegleria and that the actual amount of incorporation in the logarithmically growing populations of cells is 20 times greater than that in differentiating cells. These results suggest that nuclear DNA synthesis is reduced markedly soon after the initiation of differentiation, while cytoplasmic DNA synthesis continues. It was established from cell cycle analysis that the approximate intervals of G₁, S, G₂, and M phases were 180, 183, 90, and 28 min, respectively. Hence, the reduction in the nuclear DNA synthesis in differentiating cells is not due to the inhibition of initiation of DNA replication, but rather to the termination of the DNA replicating process. Thus DNA synthesis is curtailed in the presence of RNA and protein synthesis which are required for differentiation.     

Nuclear DNA content in differentiated tissues of sunflower (Helianthus annuus L.)

Summary Scanning cytophotometry following Feulgen-staining was used to determine nuclear DNA content in many differentiated tissues of nine cultivars, hybrids or selfed lines ofHelianthus annuus. Apart from such ephemeral tissues as endosperm and anther tapetum, it was found that tissue differentiation in sunflower occurs in the diploid condition, cells being arrested in the DNA presynthetic phase (G₁). In certain cases, however, the nuclear DNA content of differentiated G₁ cells does not exactly match the 2C DNA content found in meristematic cells, but may be either higher or lower. In endosperm and anther tapetum cells, nuclear DNA content may be as high as 24 C and 32 C, respectively. Cytological and autoradiographic analyses after³H-thymidine incorporation reveal that polyploidy in the tapetal cells is due to chromosome endoreduplication. No detectable difference between male-fertile and male-sterile plants exists as far as occurrence and level of cell polyploidy are concerned. The results are discussed in the context of previous investigations on the nuclear condition of differentiatedHelianthus annuus tissue.     

Metabolic Basis of Detoxication: Metabolism of Functional Groups : Edited by W.B. Jakoby,J.R. Bend and J. Caldwell Academic Press: New York, 1982 357 + 18 pages. $42.00

By inhibiting the α-like DNA polymerase, and therefore nuclear DNA synthesis, aphidicolin induces accumulation of suspension cultured carrot cells at the G₁/S boundary of the cell cycle. After a 24-h treatment with the drug the accumulation is complete, affecting all the cycling cells (95% of the population). Upon removal of the inhibitor, all cycling cells immediately resume nuclear DNA synthesis and move synchronously throughout the S-phase.     

Cell cycle analysis in a multinucleate green alga,Boergesenia forbesii (Siphonocladales,Chlorophyta)*

The cell cycle (nuclear division cycle) of a multinucleate green alga, Boergesenia forbesii (Harvey) Feldmann was studied using microspectrophotometry and BrdU incorporation techniques. Mitosis was observed frequently 1-4 h after the beginning of the light period, on a 16:8 h LD cycle at 25°C. Mitotic nuclei formed discrete patches. Other nuclei remained in the G₁ period. The DNA synthetic phase (S phase) was estimated to last about 12 h from microspectrophotometric study using aphidicolin inhibition just before the S phase and release from it. The G₂ period was estimated to be about 2 h, because a labeled prophase nucleus could be detected when the samples were labeled with BrdU continuously over 3 h. The incorporation pattern of BrdU changed through the S phase nucleus. In early S phase, BrdU staining was detected as many dots in the entire nucleus, while in late S phase, it was detected as several discrete regions along the nuclear membrane. Almost all nuclei in B. forbesii were in the G₁ stage after nuclear division, and the nuclei in several patches of the cell simultaneously initiated DNA synthesis. Once the nuclei entered into S phase, these nuclei continued into G₂ and mitosis. In other words, the cell cycle regulation of entrance into S phase from G₁ is an important factor in the growth and morphogenesis in B. forbesii.     

Microspectrophotometric measurements of DNA by automated computerized scanning in cycling cells of theChrysanthemum segetum shoot apex

Summary A new technique of exploitation of the data was proposed after DNA scanning microdensitometry. By using all of the measurements obtained from the seriated sections of a single nucleus, this method made it possible to estimate six characteristic parameters during the different phases of the cell cycle in the various shoot apical cells. The cells whose rate of proliferation was the highest showed the biggest variations of their nuclear and nucleolar volumes during the cell cycle. In the axial zone, where the cells have a slow cell cycle and display the longest duration of the G₁ phase, the volume occupied by dispersed DNA was greater than in the cells of the lateral zone and of the rib meristem, where the cell cycle and the G₁ phase were short. No matter what the cell type, the proportion of the dispersed and condensed DNA varied little when the G₁ and G₂ phases were compared. In the Z phase, characterized by a decondensation of the DNA, the mean DNA amount was 3.4 C. The evolution of the nuclear density during the interphase was also estimated. It is demonstrated that the main feature of the shoot apex zonation was the decondensation of the condensed DNA in the axial zone in both the G₁ and G₂ phases.     

DNA polymerase δ mediates increase in exchange production by X-radiation in human lymphocytes moving from G0 to G1

Earlier work of several laboratories established that the yields of radiation-induced ring and dicentric chromosomes are greater when human peripheral blood lymphocytes are irradiated in GH₁ some hours after phytohemagglutinin stimulation than if they are irradiated in G₀ before stimulation. Post-treatment of lymphocytes irradiated in G₀ with the DNA polymerase inhibitor aphidicolin, which is effective against both pol α and pol δ, produces a similar increase in ring and dicentric yield. We found that aphidicolin post-treatment was much less effective in increasing ring and dicentric yield increases in cells irradiated in G₁ four to five hours after stimulation. Because we had earlier found specific inhibitors of DNA pol α ineffective in producing increased yields in either G₀ or G₁ lymphocytes, we conclude that much of the G₀ to G₁ increase in yields is mediated by pol δ.     

Fructokinase is required for carbon partitioning to cellulose in aspen wood

DNA damage checkpoints delay mitotic cell‐cycle progression in response to DNA stress, stalling the cell cycle to allow time for repair. CDKB is a plant‐specific cyclin‐dependent kinase (CDK) that is required for the G₂/M transition of the cell cycle. In Arabidopsis, DNA damage leads the degradation of CDKB2, and the subsequent G₂ arrest gives cells time to repair damaged DNA. G₂ arrest also triggers transition from the mitotic cycle to endoreduplication, leading to the presence of polyploid cells in many tissues. In contrast, in rice (Oryza sativa), polyploid cells are found only in the endosperm. It was unclear whether endoreduplication contributes to alleviating DNA damage in rice (Oryza sativa). Here, we show that DNA damage neither down‐regulates Orysa;CDKB2;1 nor induces endoreduplication in rice. Furthermore, we found increased levels of Orysa;CDKB2;1 protein upon DNA damage. These results suggest that CDKB2 functions differently in Arabidopsis and rice in response to DNA damage. Arabidopsis may adopt endoreduplication as a survival strategy under genotoxic stress conditions, but rice may enhance DNA repair capacity upon genotoxic stress. In addition, polyploid cells due to endomitosis were present in CDKB2;1 knockdown rice, suggesting an important role for Orysa;CDKB2;1 during mitosis.     

Nuclear phosphoinositide 3-kinase C2β activation during G2/M phase of the cell cycle in HL-60 cells

The activity of nuclear phosphoinositide 3-kinase C2β (PI3K-C2β) was investigated in HL-60 cells blocked by aphidicolin at G₁/S boundary and allowed to progress synchronously through the cell cycle. The activity of immunoprecipitated PI3K-C2β in the nuclei and nuclear envelopes showed peak activity at 8 h after release from the G₁/S block, which correlates with G₂/M phase of the cell cycle. In the nuclei and nuclear envelopes isolated from HL-60 cells at 8 h after release from G₁/S block, a significant increase in the level of incorporation of radiolabeled phosphate into phosphatidylinositol 3-phosphate (PtdIns(3)P) was observed with no change in the level of radiolabeled PtdIns(4)P, PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃. On Western blots, PI3K-C2β revealed a single immunoreactive band of 180 kDa, whereas in the nuclei and nuclear envelopes isolated at 8 h after release, the gel shift of 18 kDa was observed. When nuclear envelopes were treated for 20 min with μ-calpain in vitro, the similar gel shift and increase in PI3K-C2β activity was observed which was completely inhibited by pretreatment with calpain inhibitor calpeptin. The presence of PI3K inhibitor LY 294002 completely abolished the calpain-mediated increase in the activity of PI3K-C2β but did not prevent the gel shift. When HL-60 cells were released from G₁/S block in the presence of either calpeptin or LY 294002, the activation of nuclear PI3K-C2β was completely inhibited. These results demonstrate the calpain-mediated activation of the nuclear PI3K-C2β during G₂/M phase of the cell cycle in HL-60 cells.     

Plastid and nuclear DNA synthesis are not coupled in suspension cells ofNicotiana tabacum

The relationship between nuclear and plastid DNA synthesis in cultured tobacco cells was measured by following³H-thymidine incorporation into total cellular DNA in the absence or presence of specific inhibitors. Plastid DNA synthesis was determined by hybridization of total radiolabeled cellular DNA to cloned chloroplast DNA. Cycloheximide, an inhibitor of nuclear encoded cytoplasmic protein synthesis, caused a rapid and severe inhibition of nuclear DNA synthesis and a delayed inhibition of plastid DNA synthesis. By contrast, chloramphenicol which only inhibits plastid and mitochondrial protein production, shows little inhibition of either nuclear or plastid DNA synthesis even after 24 h of exposure to the cells. The inhibition of nuclear DNA synthesis by aphidicolin, which specifically blocks the nuclear DNA polymeraseα, has no significant effect on plastid DNA formation. Conversely, the restraint of plastid DNA synthesis exerted by low levels of ethidium bromide has no effect on nuclear DNA synthesis. These results show that the synthesis of plastid and nuclear DNA are not coupled to one another. However, both genomes require the formation of cytoplasmic proteins for their replication, though our data suggest that different proteins regulate the biosynthesis of nuclear and plastid DNA.     

Replication and G2 checkpoints: their response to caffeine

Under long hydroxyurea treatments, evidence was obtained for the sequential activation of four checkpoints located between the onset of S phase and mitosis in Allium cepa L. root meristems. Bi-parametric flow cytometry (Br-DNA/total DNA) showed that cells initially accumulated at early S phase but, after a delay, they resumed replication and paused again at mid S phase. Cells not only overrode this second replication block but also any G₂ checkpoint they encountered. Thus, a late mitotic wave was produced in the presence of hydroxyurea. The wave was formed by cells that had apparently completed their replication (normal mitoses), while others displayed anaphases/telophases with less than the expected DNA content and with chromosomal breaks (aberrant mitoses). The presence of aberrant mitoses is direct evidence for the undue override of the two G₂ checkpoints responsible for surveillance of completion of DNA synthesis and repair, respectively. Caffeine selectively abrogated the G₂ block produced by the checkpoint that controls post-replication DNA repair, as it advanced the entry of cells into an aberrant mitosis. However, caffeine proved not to be the universal checkpoint-evading agent as postulated. Caffeine did not modify the spontaneous override of the replication checkpoints. Moreover, it seems to enforce the checkpoint that controls the completion of DNA synthesis, as the appearance of the late wave of normal mitoses produced in the presence of hydroxyurea was prevented by the use of caffeine. Received: 21 February 2000 / Accepted: 31 July 2000     

Effets d'un Sejour à l'Obscurité sur le Pouvoir Germinatif et sur L'Evolution de l'Appareil Photosynthétique des Spores de Funaria hygrometrica

In Funaria spores fine structural changes occur, especially in the plastids, during prolonged incubation in darkness on an inorganic liquid medium, although under these conditions germination cannot take place. If then exposed to illumination, these spores germinate rapidly and synchronously. Chloroplasts formed in darkness have thicker grana, absorb more CO₂, have a higher reducing power, and give a more intense paramagnetic resonance signal than chloroplasts formed in light.     

Inhibition of JNK2 and JNK3 by JNK inhibitor IX induces prometaphase arrest-dependent apoptotic cell death in human Jurkat T cells

Exposure of human Jurkat T cells to JNK inhibitor IX (JNKi), targeting JNK2 and JNK3, caused apoptotic DNA fragmentation along with G₂/M arrest, phosphorylation of Bcl-2, Mcl-1, and Bim, Δψm loss, and activation of Bak and caspase cascade. These JNKi-induced apoptotic events were abrogated by Bcl-2 overexpression, whereas G₂/M arrest, cyclin B1 up-regulation, Cdk1 activation, and phosphorylation of Bcl-2 family proteins were sustained. In the concomitant presence of the G₁/S blocking agent aphidicolin and JNKi, the cells underwent G₁/S arrest and failed to induce all apoptotic events. The JNKi-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by the Cdk1 inhibitor. Immunofluorescence microscopic analysis revealed that mitotic spindle defect and prometaphase arrest were the underlying factors for the G₂/M arrest. These results demonstrate that JNKi-induced mitochondrial apoptosis was caused by microtubule damage-mediated prometaphase arrest, prolonged Cdk1 activation, and phosphorylation of Bcl-2 family proteins in Jurkat T cells.     

Cutting edge: Chk1 directs senescence and mitotic catastrophe in recovery from G2 checkpoint arrest

Besides the well‐understood DNA damage response via establishment of G₂ checkpoint arrest, novel studies focus on the recovery from arrest by checkpoint override to monitor cell cycle re‐entry. The aim of this study was to investigate the role of Chk1 in the recovery from G₂ checkpoint arrest in HCT116 (human colorectal cancer) wt, p53^–/– and p21^–/– cell lines following H₂O₂ treatment. Firstly, DNA damage caused G₂ checkpoint activation via Chk1. Secondly, overriding G₂ checkpoint led to (i) mitotic slippage, cell cycle re‐entry in G₁ and subsequent G₁ arrest associated with senescence or (ii) premature mitotic entry in the absence of p53/p21^WAF1 causing mitotic catastrophe. We revealed subtle differences in the initial Chk1‐involved G₂ arrest with respect to p53/p21^WAF1: absence of either protein led to late G₂ arrest instead of the classic G₂ arrest during checkpoint initiation, and this impacted the release back into the cell cycle. Thus, G₂ arrest correlated with downstream senescence, but late G₂ arrest led to mitotic catastrophe, although both cell cycle re‐entries were linked to upstream Chk1 signalling. Chk1 knockdown deciphered that Chk1 defines long‐term DNA damage responses causing cell cycle re‐entry. We propose that recovery from oxidative DNA damage‐induced G₂ arrest requires Chk1. It works as cutting edge and navigates cells to senescence or mitotic catastrophe. The decision, however, seems to depend on p53/p21^WAF1. The general relevance of Chk1 as an important determinant of recovery from G₂ checkpoint arrest was verified in HT29 colorectal cancer cells.     

Model of DNA Dynamics and Replication

Before DNA replication can be initiated a definite number of adenosine triphosphate (ATP) containing pre-replication protein complexes (pre-RCs) must be assembled and bound to DNA like in a super-critical mass. A chemically driven dynamics of the Ginzburg-Landau (GL) type is derived, using the non-equilibrium equation for binding of pre-RCs to DNA and a probabilistic conformational distribution of these protein complexes. This dynamics, in which the DNA-protein system behaves like a nonlinear elastically braced string (NEBS), can control the cell cycle via conformational transitions such that G₂ cells contain exactly twice as much DNA as G₁ cells. After adjustment of previously-made derivations, the model is compared with cell growth data from the T lymphocyte MLA-144.     

In vitro DNA synthesis as indicator of mammary epithelial cell division: [14C]thymidine uptake versus flow cytometry cell cycle analysis

Summary Mammary and adipose explants from eight mid-lactation Holstein cows were co-cultured for 24 h in the presence or absence of liver explants, 1 μg/ml pituitary bovine somatotrophin, or 100 ng/ml insulinlike growth factor-I. Liver explants in the media significantly depressed DNA and protein synthesis by mammary tissue as measured by [¹⁴C]-thymidine and amino acid incorporation. As measured by flow cytometry, the concentration of DNA in the G₀G₁ and G₂M cells and the percentage of cells in the G₀G₁ population of mammary tissue was also significantly depressed by liver tissue. Changes in the percentage of cells in the S and G₂M phases were not significant. Insulinlike growth factor-I in the presence of liver explants depressed protein synthesis, thymidine incorporation, and the concentration of DNA in the G₀G₁ and G₂M cells compared to control but did not affect the percentage of cells in the G₀G₁, S, or G₂M phases. Previously it was assumed that changes in [¹⁴C]thymidine incorporation indicated that changes in cell division were occurring. Flow cytometry revealed that changes in DNA content of mammary cells as a result of liver or hormonal stimulation were not due to changes in cell division. Indications are that differences in cellular DNA content result from changes in the rate of amplification of individual genes responsible for milk protein synthesis.     

Regulation and Functional Contribution of Thymidine Kinase 1 in Repair of DNA Damage

Cellular supply of dNTPs is essential in the DNA replication and repair processes. Here we investigated the regulation of thymidine kinase 1 (TK1) in response to DNA damage and found that genotoxic insults in tumor cells cause up-regulation and nuclear localization of TK1. During recovery from DNA damage, TK1 accumulates in p53-null cells due to a lack of mitotic proteolysis as these cells are arrested in the G₂ phase by checkpoint activation. We show that in p53-proficient cells, p21 expression in response to DNA damage prohibits G₁/S progression, resulting in a smaller G₂ fraction and less TK1 accumulation. Thus, the p53 status of tumor cells affects the level of TK1 after DNA damage through differential cell cycle control. Furthermore, it was shown that in HCT-116 p53^−/− cells, TK1 is dispensable for cell proliferation but crucial for dTTP supply during recovery from DNA damage, leading to better survival. Depletion of TK1 decreases the efficiency of DNA repair during recovery from DNA damage and generates more cell death. Altogether, our data suggest that more dTTP synthesis via TK1 take place after genotoxic insults in tumor cells, improving DNA repair during G₂ arrest.     

Dovitinib induces mitotic defects and activates the G2 DNA damage checkpoint

Dovitinib (TKI258; formerly CHIR‐258) is an orally bioavailable inhibitor of multiple receptor tyrosine kinases. Interestingly, Dovitinib triggered a G₂/M arrest in cancer cell lines from diverse origins including HeLa, nasopharyngeal carcinoma, and hepatocellular carcinoma. Single‐cell analysis revealed that Dovitinib promoted a delay in mitotic exit in a subset of cells, causing the cells to undergo mitotic slippage. Higher concentrations of Dovitinib induced a G₂ arrest similar to the G₂ DNA damage checkpoint. In support of this, DNA damage was triggered by Dovitinib as revealed by γ‐H2AX and comet assays. The mitotic kinase CDK1 was found to be inactivated by phosphorylation in the presence of Dovitinib. Furthermore, the G₂ arrest could be overcome by abrogation of the G₂ DNA damage checkpoint using small molecule inhibitors of CHK1 and WEE1. Finally, Dovitinib‐mediated G₂ cell cycle arrest and subsequent cell death could be promoted after DNA damage repair was disrupted by inhibitors of poly(ADP‐ribose) polymerases. These results are consistent with the recent finding that Dovitinib can also target topoisomerases. Collectively, these results suggest additional directions for use of Dovitinib, in particular with agents that target the DNA damage checkpoint.