Different expression profiles of human cyclin B1 in normal PHA-stimulated T lymphocytes and leukemic T cells

BACKGROUND: In a previous work, we demonstrated with flow cytometry (FCM) methods that accumulation of human cyclin B1 in leukemic cell lines begins during the G(1) phase of the cell cycle (Viallard et al. , Exp Cell Res 247:208-219, 1999). In the present study, FCM was used to compare the localization and the kinetic patterns of cyclin B1 expression in Jurkat leukemia cell line and phytohemagglutinin (PHA)-stimulated normal T lymphocytes. METHODS: Cell synchronization was performed in G(1) with sodium n-butyrate, at the G(1)/S transition with thymidine and at mitosis with colchicine. Cells (leukemic cell line Jurkat or PHA-stimulated human T-lymphocytes) were stained for DNA and cyclin B1 and analyzed by FCM. Western blotting was used to confirm certain results. RESULTS: Under asynchronous growing conditions and for both cell populations, cyclin B1 expression was essentially restricted to the G(2)/M transition, reaching its maximal level at mitosis. When the cells were synchronized at the G(1)/S boundary by thymidine or inside the G(1) phase by sodium n-butyrate, Jurkat cells accumulated cyclin B1 in both situations, whereas T lymphocytes expressed cyclin B1 only during the thymidine block. The cyclin B1 fluorescence kinetics of PHA-stimulated T lymphocytes was strictly similar when considering T lymphocytes blocked at the G(1)/S phase transition by thymidine and in exponentially growing conditions. These FCM results were confirmed by Western blotting. The detection of cyclin B1 by Western blot in cells sorted in the G(1) phase of the cell cycle showed that cyclin B1 was present in the G(1) phase in leukemic T cells but not in normal T lymphocytes. Cyclin B1 degradation was effective at mitosis, thus ruling out a defective cyclin B1 proteolysis. CONCLUSIONS: We found that the leukemic T cells behaved quite differently from the untransformed T lymphocytes. Our data support the notion that human cyclin B1 is present in the G(1) phase of the cell cycle in leukemic T cells but not in normal T lymphocytes. Therefore, the restriction point from which cyclin B1 can be detected is different in the two models studied. We hypothesize that after passage through a restriction point differing in T lymphocytes and in leukemic cells, the rate of cyclin B1 synthesis becomes constant in the S and G(2)/M phases and independent from the DNA replication cycle.     

Synchronization of the cell cycle using Lovastatin

Synchronization by Lovastatin arrests many cell types reversibly in the G1 phase of the cell cycle. Here we show that Lovastatin (10 µM) mediates cell cycle arrest in human breast cancer cells, MCF-7 and MDA-MB-231, where 85% of cells accumulate in the G1 phase of the cell cycle. Addition of mevalonate (at 100X the Lovastatin concentration) releases the cells from the G1 arrest and allows for synchronous entry into late G1, S and G2/M phases of the cell cycle. The expressions of different cyclins as a marker for different phases of the cell cycle are detected by western blot analysis and indicative of synchronous transition into each of cell cycle phases following the initial G1 arrest. Due to its level of synchrony and high yield of synchronous populations of cells, Lovastatin method of cell synchronization can be used for examining gene expression patterns in a variety of different cell lines.     

Cell cycle progression in serum-free cultures of Sf9 insect cells: modulation by conditioned medium factors and implications for proliferation and productivity

Cell cycle progression was studied in serum-free batch cultures of Spodoptera frugiperda (Sf9) insect cells, and the implications for proliferation and productivity were investigated. Cell cycle dynamics in KBM10 serum-free medium was characterized by an accumulation of 50-70% of the cells in the G(2)/M phase of the cell cycle during the first 24 h after inoculation. Following the cell cycle arrest, the cell population was redistributed into G(1) and in particular into the S phase. Maximum rate of proliferation (micro(N, max)) was reached 24-48 h after the release from cell cycle arrest, coinciding with a minimum distribution of cells in the G(2)/M phase. The following declining micro(N) could be explained by a slow increase in the G(2)/M cell population. However, at approximately 100 h, an abrupt increase in the amount of G(2)/M cells occurred. This switch occurred at about the same time point and cell density, irrespective of medium composition and maximum cell density. An octaploid population evolved from G(2)/M arrested cells, showing the occurrence of endoreplication in this cell line. In addition, conditioned medium factor(s) were found to increase micro(N,max), decrease the time to reach micro(N,max), and decrease the synchronization of cells in G(2)/M during the lag and growth phase. A conditioned medium factor appears to be a small peptide. On basis of these results we suggest that the observed cell cycle dynamics is the result of autoregulatory events occurring at key points during the course of a culture, and that entry into mitosis is the target for regulation. Infecting the Sf9 cells with recombinant baculovirus resulted in a linear increase in volumetric productivity of beta-galactosidase up to 68-75 h of culture. Beyond this point almost no product was formed. Medium renewal at the time of infection could only partly restore the lost hypertrophy and product yield of cultures infected after the transition point. The critical time of infection correlated to the time when the mean population cell volume had attained a minimum, and this occurred 24 h before the switch into the G(2)/M phase. We suggest that the cell density dependent decrease in productivity ultimately depends on the autoregulatory events leading to G(2)/M cell cycle arrest.     

Screening of five specific cell cycle inhibitors using a T Cell lymphoma cell line synchrony/release assay

To obtain different cell populations at specific cell cycle stages, we used a cell culture synchronization protocol. Effects of five different cell cycle inhibitors acting throughout the cell cycle were examined by DNA flow cytometric analysis of a synchrony/release lymphoma cell line (CEM). The screening synchronized protocol showed that staurosporine, mimosine and aphidicolin are reversible G1 phase inhibitors that act at different times. Staurosporine acted in early G1, exhibited the strongest cytotoxic effect, and induced apoptosis. Mimosine and aphidicolin acted in late G1 and at the G1/S boundary, respectively. Hydroxyurea arrested CEM cells in early S phase, but later than the aphidicolin arrest point. Nocodazole synchronized CEM cells in M phase. All the inhibitors examined in this study can be used to synchronize cells at different phases of the cell cycle and were reversible with little toxicity except for staurosporine which is highly toxic. Because the regulatory mechanism of the cell cycle is disrupted by their effects on protein synthesis, however, these drugs must be used with caution.     

Opposite effect of NF-kappa B and c-Jun N-terminal kinase on p53-independent GADD45 induction by arsenite

Cell cycle checkpoint, a major genomic surveillance mechanism, is an important step in maintaining genomic stability and integrity in response to environmental stresses. Using cells derived from human bronchial epithelial cells, we demonstrate that NF-kappaB and c-Jun N-terminal kinase (JNK) reciprocally regulate arsenic trioxide (arsenite)-induced, p53-independent expression of GADD45 protein, a cell cycle checkpoint protein that arrests cells at the G(2)/M phase transition. Inhibition of NF-kappaB activation by stable expression of a kinase-mutated form of IkappaB kinase caused increased and prolonged induction of GADD45 by arsenite. In contrast, the induction of GADD45 by arsenite was transient and less potent in cells where the NF-kappaB activation pathway was normal. Analysis of the cell cycle profile by flow cytometry indicated that NF-kappaB inhibition potentiates arsenite-induced G(2)/M cell cycle arrest. Abrogation of JNK activation, on the other hand, decreased GADD45 expression induced by arsenite, suggesting a role for JNK activation in GADD45 induction. These results indicate a molecular mechanism by which NF-kappaB and JNK may differentially contribute to cell cycle regulation in response to arsenite.     

The effect of cell cycle on GFPuv gene expression in the baculovirus expression system.

The effects of cell cycle on recombinant protein production and infection yield in the baculovirus-insect cell expression system (BES) were investigated. When, at any cell cycle phase, the host cell was infected by baculovirus, the cell cycle was finally arrested at the S or G(2)/M phase with 4n DNA. In the case of G(1) or S phase-infection, cell cycle of virus-infected cells began to be arrested at S phase from 8 h post-infection or at G(2)/M phase from 4 h post-infection, respectively; while, in the case of M phase-infection, cell cycle was arrested at S phase after 12 h post-infection. When the host cell was infected at the G(1) phase, average intracellular GFPuv fluorescence intensity was 1.3-fold higher than that at G(2)/M phase at 24 h post-infection. The GFPuv expression corresponded to the profile of the G(1) cell cycle in the BES. Infection yield was measured by detection of intracellular DNA binding protein using immunohistochemical method within 7 h post-infection. The infection yield at G(1) or S phase-infection was 1.5-1.8-fold higher than that at G(2)/M phase-infection.     

The p53 pathway is synergized by p38 MAPK signaling to mediate 11,11'-dideoxyverticillin-induced G2/M arrest

The phytochemical 11,11'-dideoxyverticillin, derived from the fungus Shiraia bambusicola, has been shown to possess potent anticancer activity in vitro and in vivo. Here, we investigated the effect of 11,11'-dideoxyverticillin on cell cycle progression, and explored the potential mechanisms for this effect. A concentration- and time-dependent cell cycle blockade at G2/M phase was observed in human colon cancer cells (HCT-116) following 11,11'-dideoxyverticillin treatment and was associated with marked increases in levels of p53, phospho-p53(ser20) and phospho-Chk2(Thr 68). When wild type p53 expression was specifically inhibited by RNA interference, HCT-116 cells treated with 11,11'-dideoxyverticillin failed to arrest in G2/M and did not show increased phospho-Chk2(Thr 68). On the other hand, 11,11'-dideoxyverticillin treatment also elicited p38 MAP kinase activity and expression of phospho-p38 MAPK. Treatment with a specific p38 MAPK inhibitor (SB203580) successfully inhibited p38 MAPK and delayed the onset of G2/M arrest induced by 0.5 microM 11,11'-dideoxyverticillin after approximately 6 h, but did not abolish the induction of G2/M arrest. Additionally, SB203580 did not alter the levels of p53, phospho-p53 (ser20), or phospho-Chk2 (Thr68) proteins in 11,11'-dideoxyverticillin-treated cells. Together, these findings indicate that p53-mediated phosphorylation of Chk2 maybe plays a vital role in 11,11'-dideoxyverticillin-induced G2/M arrest, and that p38 MAPK might accelerate this progression. Our work suggests a new possibility of interactions among p53, Chk2 and p38 MAPK signaling in G2/M arrest.     

Monitoring differential synthesis of RNA in individual cells by capillary electrophoresis

A capillary electrophoresis (CE)-based technique is reported here to monitor differential RNA synthesis in individual Chinese hamster ovary cells at distinct stages of the cell proliferation cycle. Cell synchronization was achieved by the shake-off method, in which mitotic (M) cells were dislodged, and cells at G(1), S, and G(2) phases were harvested 2.5, 10, and 13 h, respectively, after synchronizing the mitotic cells. Thirty-two cells (eight from each phase) were analyzed by injecting each cell into the capillary, lysing it with dilute surfactant, separating the RNA by capillary electrophoresis, and detecting the peaks with laser-induced fluorescence. The results from single cells show that the total amount of RNA increased at each successive stage (from G(1) to M), while the relative synthetic rates of different RNA fractions varied with progression through the cycle. There was a threefold increase in the synthetic rate of total RNA from S to G(2), compared with G(1) to S. In addition, differential accumulation of specific RNA fractions was observed, with the low-molecular-mass fraction exhibiting a much higher synthetic rate from G(2) to M, relative to the rates of the larger ribosomal RNA (rRNA) fractions. Comparison of the large rRNA fractions with one another reveals that at S phase more 28S rRNA was accumulated than 18S rRNA, and at G(1) and M phases, the synthetic rate of 28S rRNA was slowed compared with that of 18S. Minimal sample preparation, combined with the separation power of CE and single-cell detection sensitivity of laser-induced fluorescence, results in a simple method for assessing differential accumulation of RNA from distinct individual cells.     

DNA replication is required To elicit cellular responses to psoralen-induced DNA interstrand cross-links

Following introduction of DNA interstrand cross-links (ICLs), mammalian cells display chromosome breakage or cell cycle delay with a 4N DNA content. To further understand the nature of the delay, previously described as a G(2)/M arrest, we developed a protocol to generate ICLs during specific intervals of the cell cycle. Synchronous populations of G(1), S, and G(2) cells were treated with photoactivated 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and scored for normal passage into mitosis. In contrast to what was found for ionizing radiation, ICLs introduced during G(2) did not result in a G(2)/M arrest, mitotic arrest, or chromosome breakage. Rather, subsequent passage through S phase was required to trigger both chromosome breakage and arrest in the next cell cycle. Similarly, ICLs introduced during G(1) did not cause a G(1)/S arrest. We conclude that DNA replication is required to elicit the cellular responses of cell cycle arrest and genomic instability after psoralen-induced ICLs. In primary human fibroblasts, the 4N DNA content cell cycle arrest triggered by ICLs was long lasting but reversible. Kinetic analysis suggested that these cells could remove up to approximately 2,500 ICLs/genome at an average rate of 11 ICLs/genome/h.     

Mitogen-activated protein kinase kinase 1-dependent Golgi unlinking occurs in G2 phase and promotes the G2/M cell cycle transition

Two controversies have emerged regarding the signaling pathways that regulate Golgi disassembly at the G(2)/M cell cycle transition. The first controversy concerns the role of mitogen-activated protein kinase activator mitogen-activated protein kinase kinase (MEK)1, and the second controversy concerns the participation of Golgi structure in a novel cell cycle "checkpoint." A potential simultaneous resolution is suggested by the hypothesis that MEK1 triggers Golgi unlinking in late G(2) to control G(2)/M kinetics. Here, we show that inhibition of MEK1 by RNA interference or by using the MEK1/2-specific inhibitor U0126 delayed the passage of synchronized HeLa cells into M phase. The MEK1 requirement for normal mitotic entry was abrogated if Golgi proteins were dispersed before M phase by treatment of cells with brefeldin A or if GRASP65, which links Golgi stacks into a ribbon network, was depleted. Imaging revealed that unlinking of the Golgi apparatus begins before M phase, is independent of cyclin-dependent kinase 1 activation, and requires MEK signaling. Furthermore, expression of the GRASP family member GRASP55 after alanine substitution of its MEK1-dependent mitotic phosphorylation sites inhibited both late G(2) Golgi unlinking and the G(2)/M transition. Thus, MEK1 plays an in vivo role in Golgi reorganization, which regulates cell cycle progression.     

Phenyl hydroquinone, an Ames test-negative carcinogen, induces Hog1-dependent stress response signaling

Recently, we have shown that phenyl hydroquinone, a hepatic metabolite of the Ames test-negative carcinogen o-phenylphenol, efficiently induced aneuploidy in Saccharomyces cerevisiae. We further found that phenyl hydroquinone arrested the cell cycle at G(1) and G(2)/M. In this study, we demonstrate that phenyl hydroquinone can arrest the cell cycle at the G(2)/M transition as a result of stabilization of Swe1 (a Wee1 homolog), probably leading to inactivation of Cdc28 (a Cdk1/Cdc2 homolog). Furthermore, Hog1 (a p38 MAPK homolog) was robustly phosphorylated by phenyl hydroquinone, which can stabilize Swe1. On the other hand, Chk1 and Rad53 were not phosphorylated by phenyl hydroquinone, indicating that the Mec1/Tel1 DNA-damage checkpoint was not functional. Mutations of swe1 and hog1 abolished phenyl hydroquinone-induced arrest at the G(2)/M transition and the cells became resistant to phenyl hydroquinone lethality and aneuploidy development. These data suggest that a phenyl hydroquinone-induced G(2)/M transition checkpoint that is activated by the Hog1-Swe1 pathway plays a role in the development of aneuploidy.     

Cycloalkyl-substituted aryl chloroethylureas inhibiting cell cycle progression in G0/G1 phase and thioredoxin-1 nuclear translocation

1-(2-Chloroethyl)-3-(4-cyclohexylphenyl)urea (cHCEU) has been shown to abrogate the presence of thioredoxin-1 into the nucleus through its selective covalent alkylation. In the present letter we have evaluated the structure-activity relationships of the substituents at positions 3 and 4 of the phenyl ring of cHCEU derivatives on cell cycle progression and thioredoxin-1 nuclear translocation. Active CEU derivatives exhibited GI(50) ranging from 1.9 to 49muM on breast carcinoma MCF-7, skin melanoma M21, and colon carcinoma HT-29 cells. On one hand, compounds 1, 2, 9c, 10c, 13, and 14 arrested the cell cycle in G(2)/M phase while CEUs 3, 4, 5c, 6c, 11c, and 12c blocked the cell division in G(0)/G(1) phase. On the other hand, CEUs 2-4, 5c, 7c, 8c, 11c, and 12c abrogated the translocation of thioredoxin-1 while the other CEU derivatives were inactive in that respect. Our results suggest that CEU substituted on the phenyl ring at position 3 or 4 by lower cycloalkyl or cycloalkoxy groups arrest cell progression in G(0)/G(1) phase through mechanism of action different from their antimicrotubule counterparts, presumably via thioredoxin-1 alkylation and modulation of its activity. The mechanism of action of these new molecules is still undetermined. However, the significant accumulation of cells in G(0)/G(1) phase suggests that these molecules may act similarly to known chemopreventive agents against cancers. In addition, the inhibition of Trx-1 nuclear localization also suggests the abrogation of an important chemoresistance mechanism towards a variety of chemotherapeutic agents.