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Quiescent cells but not cycling cells exhibit enhanced actin synthesis before they synthesize DNA.

Major proteins synthesized by Swiss 3T3 cells at different stages of the cell cycle have been analyzed using double isotope labeling and one-dimensional SDS-polyacrylamide slab gels. The synthesis of actin was previously shown to be markedly enhanced a few hours after quiescent cells initiated growth following addition of serum. In contrast, the synthesis of actin remained at a constant rate, similar to that in quiescent cells, relative to synthesis of other proteins during the entire cell cycle. We conclude that enhanced actin synthesis is a process specific for the G0 to S transit, and may serve as a marker event during this interval. In contrast, three other proteins (90,000, 57,000, and 33,000 daltons) were synthesized throughout the cell cycle at higher rates than in G0 cells, and thus, are markers characteristic of cells traversing the cell cycle. A transient increase, such as seen for actin synthesis, by cells emerging from quiescence, may represent a process that these cells must perform before they can enter the G1 portion of the cell cycle. A transient event such as this need not be a periodic event that occurs during each cycle.

Actins↗

Cell cycle control and cell division: implications for chemically induced carcinogenesis.

Eukaryotic cells proceed through an ordered series of events constituting the cell cycle, during which their chromosomes are duplicated and one copy of each daughter chromosome segregates to each daughter cell (mitosis). A precise and stringent regulation of this cell cycle is absolutely necessary for normal development of multicellular organisms; loss of cell cycle control, however, may ultimately lead to the generation of tumors. The present article provides an overview on the molecular mechanisms constituting the two most important checkpoints within the cell cycle of eukaryotic cells, that is, the spindle/mitotic checkpoint and the DNA damage checkpoint. It will be discussed how these checkpoints may be impaired by chemical carcinogens and how these interactions may contribute to the generation of aneuploidy and accumulation of somatic mutations, two major characteristics of human tumor cells.

Animals↗

Chemopreventive agent resveratrol, a natural product derived from grapes, reversibly inhibits progression through S and G2 phases of the cell cycle in U937 cells.

Resveratrol, a natural product derived from grapes, has been shown to prevent carcinogenesis in murine models. We report here that resveratrol induces antiproliferation and arrests the S phase in human histiocytic lymphoma U937 cells. Resveratrol induces arrest in the S phase at low concentrations (30-60 microM), but high concentrations do not induce S phase accumulation in U937 cells. Removal of resveratrol from the culture medium stimulates U937 cells to reenter the cell cycle synchronously, as judged by the expression patterns of cyclin E, A and by fluorescent activated cell sorting analysis. These data demonstrate that resveratrol causes S phase arrest and reversible cell cycle arrest. Thus, resveratrol provides an important new cell cycle blocker as well as a cancer chemopreventive agent.

Antineoplastic Agents, Phytogenic↗

Flow cytometry study of human cyclin B1 and cyclin E expression in leukemic cell lines: cell cycle kinetics and cell localization.

Experiments by flow cytometry (FCM) after nuclei isolation have never been done to investigate cyclins. We have conducted different experiments by FCM using whole cells and isolated nuclei to study the immunolocalization and kinetic patterns of cyclin B1 and cyclin E in various leukemic cell lines. During asynchronous growth, all whole cells had a scheduled, cell cycle phase-restricted expression of cyclin B1. By using a washless immunostaining of unfixed nuclei, cyclin B1 was detected in all cell cycle phases, including G1, although to a lesser extent than in G2/M, suggesting that in whole cells the cyclin B1 epitope is masked and accessible only in isolated nuclei. When the cells were synchronized at the G1/S boundary by thymidine or in the G1 phase by sodium n-butyrate, an identical accumulation of cyclin B1 was observed. As for cyclin E, its expression was higher with thymidine treatment than with sodium n-butyrate, particularly in nuclei. The elevated cyclin B1 level in the cells arrested at the G1/S boundary may reflect the increased half-life of this protein stabilized as the result of cyclin E overexpression. However, our FCM data also support the notion that accumulation of human cyclin B1 in leukemic cell lines begins during the G1 phase of the cell cycle, probably in the nucleus. The detection of cyclin B1 by Western blot in cells sorted in the G1 phase of the cell cycle confirms this finding. It is possible, therefore, that tumor transformation or leukemic phenotype may invariably be associated with altered cyclin B1 expression.

Blotting, Western↗

Signalling cell cycle arrest and cell death through the MMR System.

Loss of DNA mismatch repair (MMR) in mammalian cells, as well as having a causative role in cancer, has been linked to resistance to certain DNA damaging agents including clinically important cytotoxic chemotherapeutics. MMR-deficient cells exhibit defects in G2/M cell cycle arrest and cell killing when treated with these agents. MMR-dependent cell cycle arrest occurs, at least for low doses of alkylating agents, only after the second S-phase following DNA alkylation, suggesting that two rounds of DNA replication are required to generate a checkpoint signal. These results point to an indirect role for MMR proteins in damage signalling where aberrant processing of mismatches leads to the generation of DNA structures (single-strand gaps and/or double-strand breaks) that provoke checkpoint activation and cell killing. Significantly, recent studies have revealed that the role of MMR proteins in mismatch repair can be uncoupled from the MMR-dependent damage responses. Thus, there is a threshold of expression of MSH2 or MLH1 required for proper checkpoint and cell-death signalling, even though sub-threshold levels are sufficient for fully functional MMR repair activity. Segregation is also revealed through the identification of mutations in MLH1 or MSH2 that provide alleles functional in MMR but not in DNA damage responses and mutations in MSH6 that compromise MMR but not in apoptotic responses to DNA damaging agents. These studies suggest a direct role for MMR proteins in recognizing and signalling DNA damage responses that is independent of the MMR catalytic repair process. How MMR-dependent G2 arrest may link to cell death remains elusive and we speculate that it is perhaps the resolution of the MMR-dependent G2 cell cycle arrest following DNA damage that is important in terms of cell survival.

Adaptor Proteins, Signal Transducing↗

E Proteins and Id2 converge on p57Kip2 to regulate cell cycle in neural cells.

A precise balance between proliferation and differentiation must be maintained during neural development to obtain the correct proportion of differentiated cell types in the adult nervous system. The basic helix-loop-helix (bHLH) transcription factors known as E proteins and their natural inhibitors, the Id proteins, control the timing of differentiation and terminal exit from the cell cycle. Here we show that progression into S phase of human neuroblastoma cells is prevented by E proteins and promoted by Id2. Cyclin-dependent kinase inhibitors (CKI) have been identified as key effectors of cell cycle arrest in differentiating cells. However, p57Kip2 is the only CKI that is absolutely required for normal development. Through the use of global gene expression analysis in neuroblastoma cells engineered to acutely express the E protein E47 and Id2, we find that p57Kip2 is a target of E47. Consistent with the role of Id proteins, Id2 prevents activation of p57Kip2 expression, and the retinoblastoma tumor suppressor protein, a known Id2 inhibitor, counters this activity. The strong E47-mediated inhibition of entry into S phase is entirely reversed in cells in which expression of p57Kip2 is silenced by RNA interference. During brain development, expression of p57Kip2 is opposite that of Id2. Our findings identify p57Kip2 as a functionally relevant target recruited by bHLH transcription factors to induce cell cycle arrest in developing neuroblasts and suggest that deregulated expression of Id proteins may be an epigenetic mechanism to silence expression of this CKI in neural tumors.

Adenoviridae↗

Intracellular localization of cyclin B1 during the cell cycle in glioma cells.

We investigated cyclin B1 expression during the cell cycle in human glioma cells cultured under asynchronous growing condition by two cytometry techniques: flow cytometry (FCM) and laser scanning cytometry (LSC). FCM analysis revealed the specific accumulation of cyclin B1 in G2/M phase with a wide intercellular variation (Dunphy WG: Trend Cell Biol 4:202-207, 1994). It is noteworthy that LSC, which is characterized by rapid quantitative analysis followed by imaging, allows morphological observation of the intracellular distribution of cyclin B1 as a function of cell cycle position cell by cell (Hunter T: Cell 75:839-841, 1993). Cyclin B1 was virtually undetectable in cells from G0/G1 phase to mid S phase, but became visible in the cytoplasm in late S phase. As cells proceeded within G2 phase, the level of cyclin B1 rapidly increased in the perinuclear region of the cytoplasm, but cyclin B1 was still faintly present in the nucleus. Cyclin B1 appeared in the nucleus at the mitotic phase. Then the nuclear membrane was disrupted and cyclin B1 was distributed evenly in the cell. The level of cyclin B1 was maximum in metaphase. However, it abruptly degraded at the end of metaphase, and subsequently G1 cells were cyclin B1 negative.

Cell Cycle↗

Trichostatin A regulates hGCN5 expression and cell cycle on Daudi cells in vitro.

The expression of human general control of amino acid synthesis protein 5 (hGCN5) in human Burkitt's lymphoma Daudi cells in vitro, effects of Trichostatin A (TSA) on cell proliferation and apoptosis and the molecular mechanism of TSA inhibiting proliferation of Daudi cells were investigated. The effects of TSA on the growth of Daudi cells were studied by 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. The effect of TSA on the cell cycle of Daudi cells was assayed by a propidium iodide method. Immunochemistry and Western blot were used to detect the expression of hGCN5. The proliferation of Daudi cells was decreased in TSA-treated group with a 24 h IC50 value of 415.3979 microg/L. TSA induced apoptosis of Daudi cells in a time- and dose-dependent manner. Treatment with TSA (200 and 400 microg/L) for 24 h, the apoptosis rates of Daudi cells were (14.74+/-2.04) % and (17.63+/-1.25) %, respectively. The cell cycle was arrested in G0/G1 phase (50, 100 microg/L) and in G2/M phase (200 microg/L) by treatment with TSA for 24 h. The expression of hGCN5 protein in Daudi cells was increased in 24 h TSA-treated group by immunochemistry and Western blot (P<0.05). It was suggested that TSA as HDACIs could increase the expression of hGCN5 in Daudi cells, and might play an important role in regulating the proliferation and apoptosis of B-NHL cell line Daudi cells.

Apoptosis↗

Expression and roles of Cl- channel ClC-5 in cell cycles of myeloid cells.

ClC-5 is a chloride channel known to be expressed in the kidney. We previously reported that ClC-5 mRNA was also strongly expressed in immature human myeloid cell line (HL-60), but weakly expressed in mature neutrophils. To clarify the underlying mechanisms, we examined the relationship between ClC-5 expression and cell cycle. Dimethyl sulfoxide treatment of HL-60 that causes differentiation with G0/G1 cell cycle arrest decreased the expression of ClC-5 mRNA. Cell sorting and synchronization experiments revealed that ClC-5 mRNA expression was high in S and G2/M phases and low in G0/G1 phase. ClC-5 antisense oligonucleotide suppressed proliferation of HL-60 cells with a decrease in ClC-5 protein expression, probably due to G2 arrest. These results suggest that cell cycle-dependent expression of ClC-5 has a role in cell cycle progression in myeloid cells.

Cell Cycle↗

Conservative homologous recombination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells.

DNA double-strand breaks (DSBs) are repaired by either homologous recombination (HR) or non-homologous end joining (NHEJ) in mammalian cells. Repair with NHEJ or HR using single-strand annealing (SSA) often results in deletions and is generally referred to as non-conservative recombination. Error-free, conservative HR involves strand invasion and requires a homologous DNA template, and therefore it is generally believed that this type of repair occurs preferentially in the late S, G2 and M phases of the cell cycle, when the sister chromatid is available. There are several observations supporting this hypothesis, although it has not been tested directly. Here, we synchronize human SW480SN.3 cells in the G1/G0 (with serum starvation), S (with thymidine block) and M (with nocodazole) phases of the cell cycle and investigate the efficiency of conservative HR repair of an I-SceI-induced DSB. The frequency of HR repair of DSBs was 39 times higher in S-phase cells than in M-phase cells and 24-fold higher than in G1/G0 cells. This low level of conservative HR occurs even though a homologous template is present within the recombination substrate. We propose that this can be explained by an absence of recombination proteins outside the S phase or alternatively that there maybe factors that suppress HR in G1/G0 and M. Furthermore, we found that HR repair of DSBs involves short tract gene conversion in all the phases of the cell cycle. This indicates that the same pathway for conservative HR is employed in the repair of DSBs regardless of phase of the cell cycle and that only the frequency is affected.

Cell Cycle↗

Transient ectopic expression of PTEN in thyroid cancer cell lines induces cell cycle arrest and cell type-dependent cell death.

The tumour suppressor gene PTEN/MMAC1/TEP1 has been implicated in a variety of human cancers and several inherited hamartoma tumour syndromes, including Cowden syndrome, which has a high risk of breast and thyroid cancer. We have previously reported that overexpression of PTEN in MCF-7 breast cancer cells induces cell cycle arrest and apoptosis. In this study, we analysed PTEN status at both the structural and expression levels and explored PTEN's growth-suppressive effects on thyroid. We found that 1 of 10 thyroid cancer lines [follicular thyroid carcinoma FTC-133] had hemizygous deletion and a splice variant IVS4--19G-->A in the remaining allele. Four lines, including FTC-133, express PTEN mRNA at low levels. In general, PTEN protein levels correlated with mRNA levels, except for NPA87, which has low levels of transcript and relatively high levels of PTEN protein. Transient expression of PTEN in seven thyroid cancer cell lines resulted in G(1) arrest in two well differentiated papillary thyroid cancer lines (PTCs) and both G(1) arrest and cell death in the remaining five lines, including three FTCs, one poorly differentiated PTC and one undifferentiated thyroid cancer. The level of phosphorylated Akt was inversely correlated with the endogenous level of PTEN protein and overexpression of PTEN-blocked Akt phosphorylation in all cells analysed. Our results suggest that downregulation of PTEN expression at the mRNA level plays a role in PTEN inactivation in thyroid cancer and PTEN exerts its tumour-suppressive effect on thyroid cancer through the inhibition of cell cycle progression alone or both cell cycle progression and cell death.

Adenocarcinoma, Follicular↗

Expression of integrin beta1 and its roles on adhesion between different cell cycle hepatocellular carcinoma cells (SMMC-7721) and human umbilical vein endothelial cells.

To investigate expression of integrin beta1 and its roles on adhesion between different cell cycle hepatocellular carcinoma cell (HCC) and human umbilical vein endothelial cells (HUVEC), the synchronous G1 and S phase HCC were achieved through thymine-2-deoxyriboside and colchicines sequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Expression of integrin beta1 on hepatocellular carcinoma cells was detected with flow cytometer. Further, the adhesive force of HCC to HUVEC and the role of integrin beta1 in this adhesive course were studied by micropipette aspiration technique. The results showed that percentage of each cyclic phases of the controlled HCC (non-synchronous) are: G2 + M phase, 11%; G1 phase, 54%; S phase, 36%; the synchronous rates of G1 and S phase HCC amount to 74 and 98%, respectively. The expressive fluorescent intensity of integrin beta1 in G1 phase HCC is depressed significantly than the values of S phase and controlled HCC. Accordingly, the adhesive forces of G1 phase HCC to HUVEC was significantly lower than the value of S phase cells (P < 0.01), but it has no remarkable difference when compared the adhesive force values of S phase HCC with control; the contribution of integrin beta1 was about 50% in the adhesion of HCC to HUVEC. It suggested that HCC would be synchronized preferably in G1 and S phase with thymine-2-deoxyriboside and colchicines, the adhesive molecule integrin beta1 expressed in a high lever in HCC and presented differences in vary cell cycle, and integrin beta1 played an important roles in adhesion of HCC to HUVEC. Possibly, S phase HCC take a great action in this adhesive course.

Antibodies, Monoclonal↗

Cell cycle specificity of cytogenetic damage induced by 3,4-epoxy-1- butene.

3,4-epoxy-1-butene (EB), a primary metabolite of butadiene, is a direct-acting "S-dependent" genotoxicant that can induce sister chromatid exchanges (SCEs) and chromosome aberrations (CAs) in cycling cells in vitro. However, EB is almost inactive when splenic or peripheral blood lymphocytes are exposed at the G(0) stage of the cell cycle. To investigate whether repair of DNA lesions is responsible for the lack of cytogenetic responses seen after G(0) treatments, we used cytosine arabinoside (ara-C) to inhibit DNA polymerization during DNA repair. If enough repairable lesions are present, double-strand breaks should accumulate and form chromosome-type ("S-independent") deletions and exchanges. This is exactly what occurred. EB induced chromosome deletions and dicentrics at the first division following treatment, when the EB exposure was followed by ara-C. Without ara-C treatment, there was no induction of CAs. These experiments indicate that the relatively low levels of damage induced by EB in G(0) lymphocytes are removed by DNA repair prior to DNA synthesis and thus, before the production of SCEs or chromatid-type aberrations.

Cell Cycle↗

The cell cycle.

The cell cycle and the cell cycle control system are the engines that drive life. They allow for the processes of cell renewal and the growth of organisms, under controlled conditions. The control system is essential for the monitoring of normal cell growth and replication of genetic material and to ensure that normal, functional daughter cells are produced at completion of each cell cycle. Although certain clinical applications exist which take advantage of the events of the cell cycle, our understanding of its mechanisms and how to manipulate them is infantile. The next decades will continue to see the effort of many researchers focused upon unlocking the mysteries of the cell cycle and the cell cycle control system.

Animals↗

[The kinetics of the transition to DNA synthesis in the cell cycle of sister cells of the ESK line in culture].

The proliferation kinetics of a cultured pig embryo kidney cell line, PK, was studied by time lapse cinemicrography and 3H-TdR autoradiography. The duration and variability of all phases of the cell cycle was estimated. Evidence is presented that the variation in the cell cycle transit time of both unrelated and sibling cells results mainly from the variation in transit of G1-phase. These results indicate that the kinetics of the entry of cells into the S-phase represents the first order kinetics and does not contradict the transition probability model of cell cycle control. Analysis of the labeling pattern of sister cells reveals a clear correlation of the sister cells G1 transit time.

Animals↗

Immunocytochemical analysis of the coiled body in the cell cycle and during cell proliferation.

Coiled bodies (CBs) are small, round structures found in the nucleoplasm of most eukaryotic cells. Human autoantibodies to a 80-kDa protein, p80-coilin, are immunohistologic markers for CBs. A polyclonal rabbit antiserum (R288) raised against recombinant p80-coilin was shown to have similar immunochemical properties as human autoantibodies and was used to analyze the expression of p80-coilin-associated CBs in cell cultures synchronized by double thymidine block, nocodazole arrest, serum starvation, or hormonal deprivation. By employing thymidine block and nocodazole arrest of HeLa cells, CBs were observed in immunofluorescent studies to be largest in size in the S and G2 phases of the cell cycle. These large CBs might have coalesced into one or two such structures per cell from smaller and more numerous CBs of three to eight per cell during the mid G1 phase of the cell cycle. No CB-like structures were observed in mitosis and early G1. However, immunoblotting analyses showed that the total amount of p80-coilin remained approximately the same throughout the cell cycle. When HeLa cells were separated into soluble and particulate fractions, p80-coilin was detected predominantly in the soluble fraction in mitosis and early G1, whereas it was present predominantly in the particulate fraction in late G1, S, and G2 when structurally distinct CBs were observed. In the analysis of CBs in two experimental models of cell proliferation (reversal of 3T3 serum starvation and FRTL-5 thyrotropin deprivation), proliferating cells contained larger, brighter, and more numerous CBs as well as a > 2-fold increase in the total amount of p80-coilin compared to that in quiescent cells. The expression of p80-coilin in quiescent cells induced to proliferate and the cyclic formation and breakdown of CBs might be consistent with the notion that CBs may be specialized centers related to the maturation of mRNA, but this evidence is indirect and needs further definitive study.

Autoantigens↗

Activation of the A3 adenosine receptor affects cell cycle progression and cell growth.

The A3 adenosine receptor has been implicated in modulation of cell growth. As a first step to the characterization of the underlying mechanisms, we exposed Chinese hamster ovary (CHO) cells transfected with the human A3 receptor (A3R-CHO) to selective A3 receptor ligands. At micromolar concentrations, the A3 agonists N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) and its 2-chloro derivative Cl-IB-MECA reduced cell number, with no effects on either parental CHO cells (not expressing any adenosine receptor), or CHO cells transfected with the human A1 receptor. Cl-IB-MECA also reduced cell number in the human HEK293 cell line transfected with the human A3 receptor cDNA as opposed to the respective untransfected wild-type cells. In A3R-CHO, agonist-induced effects were antagonized by nanomolar concentrations of A3 antagonists, including the triazoloquinazoline derivative MRS 1220, the dihydropyridine derivative MRS 1191, and the triazolonaphthyridine derivative L-249,313. A3 agonist-induced effects were not due to modulation of cell adhesion, nor to necrosis or apoptosis. Growth curves revealed highly impeded growth, and flow-cytometric analysis showed markedly reduced bromodeoxyuridine incorporation into nuclei. The effect on cell cycle was completely antagonized by MRS1191. Hence, activation of the human A3 receptor in A3R-CHO results in markedly impaired cell cycle progression, suggesting an important role for this adenosine receptor subtype in cell cycle regulation and cell growth.

Adenosine↗

[Cell cycles of cancer cells and chemotherapy--flow cytometric observation of the effects of chemotherapeutic agents on cell kinetics of cultured human lung cancer cells].

The necessity to assess the influence of chemotherapeutic agents on cell cycle kinetics has been stressed for development of effective cancer chemotherapy. Effects of these agents on cell cycle progression in cultured human lung cancer cells, PC-3 (adeno) and PC-6 (small cell), were studied by flow cytometry (FCM) with computer analysis. DNA distributions of the cells stained with propidium iodide were expressed numerically. Firstly the influence of adriamycin (ADR), then methotrexate (MTX), and finally, sequential combined chemotherapy with ADR and MTX on cell cycle kinetics were examined. The results are summarized as follows. Exposure to ADR induced concentration dependent cell killing effect in PC-3 and PC-6, while exposure to MTX showed mainly time dependent cell growth inhibition. By FCM, difference of effect of ADR between those two cell types was certified. Namely, in PC-3 low ADR concentration lead to G2 accumulation with decrease of G0/G1, polyploization, and high ADR concentration to S accumulation due to S-G2/M delay. Furthermore, in the highest ADR concentration (10 micrograms/ml), G1, G1-S boundary accumulation were observed. On the contrary, exposure of PC-6 to ADR remained to show only G2 accumulation with a slight decrease of G0/G1. Exposure of PC-6 to MTX lead to G2/M decrease with delay of G0/G1-S progression followed by S accumulation which was ascribed to delay of S-G2/M progression. Sequential combined chemotherapy showed superiority of MTX-ADR to ADR-MTX and other regimens in cytocidal effect, and the difference might be attributable to induction of partial synchronization to G0/G1-S boundary with MTX exposure. It was emphasized that FCM is a indispensable method for analysis of the influence of chemotherapeutic agents on cell cycle kinetics.

Adenoma↗