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PubMed2002

Early progression from dimethyl sulfoxide-induced G(0)/G(1) arrest in L(1210) cells.

Recently, dimethyl sulfoxide (DMSO) has been used as a convenient cryoprotectant for stem cells in stem cell transplantation using allogenic peripheral blood or umbilical cord blood. As the stem cells have a multipotency, clarification of the extent of cell proliferation after transplantation is difficult. In the present study, DMSO gradually induced G(0)/G(1) arrest in mouse leukemia L(1210) cells with good cell viability. After removal of DMSO, the cells proliferated appropriately, resulting in expression of the DNA-synthesizing enzymes thymidylate synthase and thymidine kinase within 6h, and the cells entering into S phase within 12h. The sequence was followed by the marked activation of both enzymes within 24h and the increase of bromodeoxyuridine (BrdU) immunoreactive (S phase) cells with rapid cell proliferation within 36 h. In conclusion, mouse leukemia L(1210) cells, which were treated with 1.5% DMSO for 96 h, tolerated the treatment and reversed the cell cycle arrest within 36 h.

Animals↗
PubMed2003

Flow cytometric analysis of the cell cycle in different coconut palm (Cocos nucifera L.) tissues cultured in vitro.

We conducted a study of the cell cycle of coconut palm tissues cultured in vitro in order to regulate regeneration. Coconut palm is a plant for which it is difficult to monitor the ability of the meristematic cells to actively divide. Cell nuclei were isolated from various types of coconut palm tissues with and without in vitro culture. After the nuclei were stained with propidium iodide, relative fluorescence intensity was estimated by flow cytometry. Characterization of the cell cycle reinforced the hypothesis of a block in the G(0)/G(1) and G(1)/S phases of the coconut cells. A time-course study carried out on immature leaves revealed that this block takes place gradually, following the introduction of the material in vitro. Synchronization of in vitro-cultured leaves cells using 60 micro M aphidicholin revealed an increase in the number of nuclei in the S phase after 108 h of treatment. The significance of these results is discussed in relation with the ability of coconut tissue cultured in vitro to divide.

Aphidicolin↗
PubMed1991

A general method for modeling cell populations undergoing G1----G0 transitions during development.

The transition from the dividing state to a non-dividing, terminally differentiated state is common to the history of most populations of cells during development. Quantifying such transitions and events related to them is often difficult, even in those cases for which there is a good tissue culture model, because the process is asynchronous and occurs against a background of continued extensive growth. A general model for analyzing these complex population changes is presented here. In the absence of definitive data, the model provides projections of the possible range, under a given set of boundary values, for the rate of terminal differentiation, the overall growth rate, and the degree of cell death. On the other hand, given data on the rate of DNA accumulation, dividing cell fraction, and generation time, the model provides the effective partitioning coefficient between the dividing and non-dividing states averaged over the population, at a given time. These data also allow for an assessment of the degree of actual cell death against a background in which significant numbers of cells are withdrawing from the cell cycle. The types of data required with respect to the model's ability to resolve the nature of a G0 transition "window" within the cell cycle are also discussed.

Animals↗
PubMed1995

Evidence for the organization of chromatin in megabase pair-sized loops arranged along a random walk path in the human G0/G1 interphase nucleus.

We determined the folding of chromosomes in interphase nuclei by measuring the distance between points on the same chromosome. Over 25,000 measurements were made in G0/G1 nuclei between DNA sequences separated by 0.15-190 megabase pairs (Mbp) on three human chromosomes. The DNA sequences were specifically labeled by fluorescence in situ hybridization. The relationship between mean-square interphase distance and genomic separation has two linear phases, with a transition at approximately 2 Mbp. This biphasic relationship indicates the existence of two organizational levels at scales > 100 kbp. On one level, chromatin appears to be arranged in large loops several Mbp in size. Within each loop, chromatin is randomly folded. On the second level, specific loop-attachment sites are arranged to form a supple, backbonelike structure, which also shows characteristic random walk behavior. This random walk/giant loop model is the simplest model that fully describes the observed large-scale spatial relationships. Additional evidence for large loops comes from measurements among probes in Xq28, where interphase distance increases and then locally decreases with increasing genomic separation.

Cell Cycle↗
PubMed1991

Analysis of cell cycle kinetics using flow cytometry from paraffin-embedded tissues in endometrial adenocarcinoma.

Flow cytometric analysis of cell cycle kinetics from paraffin-embedded tissues was performed to evaluate the DNA heterogeneity and risk factor of endometrial adenocarcinoma. We studied 54 tumor samples and 6 controls with normal endometrium, and all samples were measured for S-phase fraction (SPF) and proliferative index (PI). Intratumor variations of SPF and PI values were evaluated at 3 different sites of the tumor; (1) primary endometrial lesion, (2) the site of myometrial invasion, and (3) metastatic lesions. SPF and PI values of endometrial adenocarcinoma were significantly higher than those of normal endometrium. There was a significant correlation between the PI value and the histological grade. SPF and PI values at the primary endometrial lesion had a significant correlation with the prognosis of this cancer at stage III or IV. SPF and PI values which were measured at the site of myometrial invasion tended to be lower than those from other sites. Our results indicate that the SPF and PI values of endometrial lesions are one of the important prognostic factors of endometrial adenocarcinoma at stage III or IV. In addition, DNA syntheses of endometrial adenocarcinoma cells display intratumor heterogeneity.

Adenocarcinoma↗
PubMed1998

Rapamycin induces the G0 program of transcriptional repression in yeast by interfering with the TOR signaling pathway.

The macrolide antibiotic rapamycin inhibits cellular proliferation by interfering with the highly conserved TOR (for target of rapamycin) signaling pathway. Growth arrest of budding yeast cells treated with rapamycin is followed by the program of molecular events that characterizes entry into G0 (stationary phase), including the induction of polymerase (Pol) II genes typically expressed only in G0. Normally, progression into G0 is characterized by transcriptional repression of the Pol I and III genes. Here, we show that rapamycin treatment also causes the transcriptional repression of Pol I and III genes. The down-regulation of Pol III transcription is TOR dependent. While it coincides with translational repression by rapamycin, transcriptional repression is due in part to a translation-independent effect that is evident in extracts from a conditional tor2 mutant. Biochemical experiments reveal that RNA Pol III and probably transcription initiation factor TFIIIB are targets of repression by rapamycin. In view of previous evidence that TFIIIB and Pol III are inhibited when protein phosphatase 2A (PP2A) function is impaired, and that PP2A is a component of the TOR pathway, our results suggest that TOR signaling regulates Pol I and Pol III transcription in response to nutrient growth signals.

Antifungal Agents↗
PubMed2001

Cord blood G(0) CD34+ cells have a thousand-fold higher capacity for generating progenitors in vitro than G(1) CD34+ cells.

We examined the functional differences between G(0) and G(1) cord blood CD34+ cells for up to 24 weeks in serum-free suspension culture, containing Flt-3 ligand, thrombopoietin and stem cell factor. By week 24, there is more than a 1,000-fold difference in granulocyte, macrophage-colony-forming cells (GM-CFC) cumulative production between the two populations, with cultures initiated from G(0) demonstrating an amplification of 1.1 x 10(5)-1.8 x 10(6) of GM-CFC compared to 45-2.7 x 10(3) for the G(1) cells. Cells from the initial G(0) population are able to produce about 250-fold higher numbers of BFU-E than those from G(1) which translates to 3 x 10(3)-1.1 x 10(5)-fold expansion and 25-390-fold expansion for G(0) and G(1), respectively. This amplification of the progenitor cells is reflected in finding that a greater proportion of the progeny of the G(0) population are CD34+, resulting in a 600-fold expansion of CD34+ cells at week 8. As in other in vitro systems, total cell expansion is less discriminatory of stem cell behavior than progenitor cells, and there is no significant difference in total cell numbers between G(0) and G(1) cultures with a mean fold expansion of 2 x 10(7) at 24 weeks.

Antigens, CD34↗
PubMed2006

Furosemide, a blocker of Na+/K+/2Cl- cotransporter, diminishes proliferation of poorly differentiated human gastric cancer cells by affecting G0/G1 state.

Furosemide, a blocker of Na(+)/K(+)/2Cl(-) cotransporter (NKCC), is often used as a diuretic to improve edema, ascites, and pleural effusion of patients with cancers. The aim of the present study was to investigate whether an NKCC blocker affects cancer cell growth. If so, we would clarify the mechanism of this action. We found that poorly differentiated gastric adenocarcinoma cells (MKN45) expressed the mRNA of NKCC1 three times higher than moderately differentiated ones (MKN28) and that the NKCC in MKN45 showed higher activity than that in MKN28. A cell proliferation assay indicates that furosemide significantly inhibited cell growth in MKN45 cells, but not in MKN28 cells. Using flow cytometrical analysis, we found that the exposure to furosemide brought MKN45 cells to spend more time at the G(0)/G(1) phase, but not MKN28 cells. Based on these observations, we indicate that furosemide diminishes cell growth by delaying the G(1)-S phase progression in poorly differentiated gastric adenocarcinoma cells, which show high expression and activity of NKCC, but not in moderately differentiated gastric adenocarcinoma cells with low expression and NKCC activity.

Adenocarcinoma↗
PubMed2003

Caspase activity is required for stimulated B lymphocytes to enter the cell cycle.

Following activation with proliferative stimuli, including ligation of CD40, dense human tonsillar B cells (>98% cells in G(0)) have increased cleavage and activation of caspase-8 and -6 accompanied by decreased caspase-3 activation and apoptosis. Proliferation was blocked by either a broad specificity caspase inhibitor or inhibitors selective for caspase-6 or caspase-8. In contrast, an inhibitor selective for caspase-3 was without effect. Furthermore, induction of cyclin D and cyclin-dependent kinase 4 mRNA and protein was blocked upon inhibition of caspase-6, but not caspase-3. Thus, caspase-6-like activity is required for quiescent B cells to increase the expression of genes required for entry into G(1). In support of this model, the transcriptional suppressor special AT-rich sequence-binding protein 1, a preferred caspase-6 substrate, was cleaved upon B cell stimulation. Caspase activity was not required for all signaling events, as caspase inhibitors did not affect the phosphorylation of p42/44 mitogen-activated protein kinase, the expression of the survival factor cellular inhibitor of apoptosis 2, or the production of IL-6 by stimulated G(0) B cells. These findings suggest a mechanism by which caspase-6 may selectively allow entry of quiescent B cells into the cell cycle.

Amino Acid Chloromethyl Ketones↗
PubMed1994

Requirement of casein kinase 2 for entry into and progression through early phases of the cell cycle.

Requirement of protein kinase CK2 during cell cycle was examined by specific perturbation of CK2 in the intact cell by antisense-oligodeoxynucleotides and microinjection of antibodies. When quiescent human primary lung fibroblasts (IMR-90) were exposed before growth stimulation to oligodeoxynucleotides complementary to the translation start region of mRNAs encoding subunit alpha or beta, a significant inhibition of growth stimulation by epidermal growth factor or serum was observed. The inhibition was reversible and decreased or abolished with mutated antisense-oligodeoxynucleotides. The inhibitory effect coincided with a decrease of CK2 protein (immunostaining with beta subunit antibody) at entry into and during the first several hours of the cell cycle. Injection of beta-specific monoclonal and polyclonal antibodies into IMR-90 cells caused significant inhibition of growth stimulation. The inhibition was reversible, not observed with control antibodies, and strongly reduced by coinjection of CK2 holoenzyme. Cytoplasmic injection inhibited up to 50-60% and was effective at two intervals within the first 2 h and at 12-16 h poststimulation, i.e., at G0/G1 phase transition and at G1/S boundary, respectively. The inhibition at G0/G1 transition is paralleled by an inhibition of cytoplasmic-nuclear translocation of beta subunit protein. Injection of beta antibodies into the nucleus inhibited growth stimulation by as much as 80-85% and was effective for the first 6 h poststimulation, i.e., at G0/G1 phase transition and progression through the adjoining early G1 phase. Nuclear as well as cytoplasmic injections performed during S phase affected neither DNA synthesis nor cell division.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1995

The replication in vitro of the gammaherpesvirus bovine herpesvirus 4 is restricted by its DNA synthesis dependence on the S phase of the cell cycle.

Because several observations have suggested that replication of the gammaherpesvirus bovine herpesvirus 4 (BHV-4) is influenced by the physiological state of the host cell, a study was carried out to determine the relationship between BHV-4 infection and the cell cycle. The temporal expression of BHV-4 late (L) proteins in unsynchronized cell cultures was first investigated by flow cytometry. Interestingly, L protein expression occurred in a limited number of cells infected with a high multiplicity of infection, and a reciprocal correlation between the percentage of positive cells and the cell density at the time of infection was demonstrated. Moreover, the finding that a BHV-4 early-late protein was expressed in nearly all the cells suggested that a blockage in the viral replication cycle occurred in some infected cells at the stage of viral DNA synthesis or L protein expression. Because this blockage could be the consequence of the dependence of one or both of these events on the cell cycle, they were investigated after infection of synchronized cell cultures. The following findings were made. (i) Cell transition through the S phase quantitatively increased the rate of BHV-4 DNA replication. (ii) BHV-4 DNA synthesis could not be detected in cells arrested in G0. (iii) Synchronization of MDBK cells with Lovastatin before infection increased the percentage of cells expressing L proteins. (iv) In contrast, infection of cells arrested in G0 led to few positive cells. Taken together these results showed that BHV-4 DNA replication and consequently the expression of L proteins are dependent on the S phase of the cell cycle. This dependence could be of importance for several biological properties of BHV-4 infection in vitro and might have implications for the biology of the virus in vivo.

Animals↗
PubMed1997

Mitogenic stimulation of resting T cells causes rapid phosphorylation of the transcription factor LSF and increased DNA-binding activity.

The mammalian transcription factor LSF (CP2/LBP-1c) binds cellular promoters modulated by cell growth signals. We demonstrate here that LSF-DNA-binding activity is strikingly regulated by induction of cell growth in human peripheral T lymphocytes. Within 15 min of mitogenic stimulation of these cells, the level of LSF-DNA-binding activity increased by a factor of five. The level of LSF protein in the nucleus remained constant throughout this interval. However, a rapid decrease in the electrophoretic mobility of LSF, attributable to phosphorylation, correlated with the increase in DNA-binding activity. pp44 (ERK1) phosphorylated LSF in vitro on the same residue that was phosphorylated in vivo, specifically at amino acid position 291, as indicated by mutant analysis. As direct verification of the causal relationship between phosphorylation and DNA-binding activity, treatment in vitro of LSF with phosphatase both increased the electrophoretic mobility of the protein and decreased LSF-DNA-binding activity. This modulation of LSF-DNA-binding activity as T cells progress from a resting to a replicating state reveals that LSF activity is regulated during cell growth and suggests that LSF regulates growth-responsive promoters.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed2003

HCV core/gC1qR interaction arrests T cell cycle progression through stabilization of the cell cycle inhibitor p27Kip1.

Hepatitis C virus (HCV) is efficient in the establishment of persistent infection. We have previously shown that HCV core protein inhibits T cell proliferation through its interaction with the complement receptor, gC1qR. Here we show that HCV core-induced inhibition of T cell proliferation involves a G(0)/G(1) cell cycle arrest, which is reversible upon addition of anti-gC1qR antibody. Correspondingly, the expression of cyclin-dependent kinases (Cdk) 2/4 and cyclin E/D, as well as subsequent phosphorylation of retinoblastoma (pRb), is reduced in core-treated T cells in response to mitogenic stimulation. Remarkably, degradation of p27(Kip1), a negative regulator of both Cdk4/cyclin D and Cdk2/cyclin E complexes, is significantly diminished in T cells treated with HCV core upon mitogenic stimulation. These data indicate that the stability of p27(Kip1) by HCV core is associated with blocking activated T cells for the G(1) to S phase transition and inhibiting T cell proliferation.

Carrier Proteins↗
PubMed1996

Antagonistic actions of phorbol ester in mammalian G0-->G1-->S cell cycle transition.

We have developed a protocol that reveals two antagonistic effects of phorbol-12-myristate-12-acetate (PMA) on the G0-->G1-->S transition of mammalian cell cycle. Balb-3T3 (Clone A31) cells arrested in G0 by serum starvation can be stimulated to traverse the G1 phase and initiate DNA synthesis 12 h later by a 2-h pulse with PMA. In contrast with this early stimulatory effect, PMA has an inhibitory effect when presented to the cells during the last 6 h of G1. PMA is able to inhibit DNA synthesis initiation irrespective of the triggering agent, i.e., serum, fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, or PMA itself (presented as an early pulse). We have established that the critical period for the PMA inhibitory effect is between 6 and 8 h after cell stimulation. This dual effect of PMA is not a peculiarity of Balb-3T3 (clone A31) cells because it is also observed with other fibroblastic cell lines, namely, SWISS 3T3, NIL 8, and RAT 1, and also with the epithelial Y-1 adrenocortical cell line. Treatment with PMA for 0.5 or 2 h activates protein kinase C (PKC) in Balb-3T3-A31 cells, but is not sufficient to down-regulate the enzyme because a second 30-min PMA pulse applied between 6 and 6.5 h activates PKC again. On the other hand, a continuous 6.5-h PMA treatment causes PKC down-regulation; therefore, the inhibitory effect of PMA could be mediated by PKC. Growth factor early response proto-oncogenes c-myc, c-fos, and c-jun are induced transiently by both early and late PMA pulses, suggesting that these genes are not involved in the PMA inhibitory effect.

3T3 Cells↗
PubMed1999

Transduction of human progenitor hematopoietic stem cells by human immunodeficiency virus type 1-based vectors is cell cycle dependent.

Human immunodeficiency virus (HIV) type 1 vectors are highly efficient in their ability to transduce human progenitor hematopoietic stem cells (PHSC). Although mitosis was not required for transduction of these cells, transduction rates were much greater once cells had been cultured in the presence of cytokines. Transduction rates, however, rarely exceeded 70%. We demonstrate here that there is a distinct subpopulation that is more easily transduced by HIV vectors. These cells were distinguished by a disproportionate population in the S/G2/M phases of the cell cycle. By sorting them prior to transduction, we found that those cells in either the G1 or S/G2/M fraction were more readily transduced than G0 cells. Maintaining the cells in G0 by omitting cytokines from the medium reduced transduction rates by up to 10-fold. Addition of cytokines to the medium immediately after transduction did not improve the transduction efficiency as measured by expression of the transgene. Analysis of replication intermediates indicated that the block to transduction of G0 cells operated near the time of initiation of reverse transcription. These results suggest that although lentivirus vectors can transduce nondividing PHSC, transduction efficiency is severalfold greater once the cells exit G0 and enter G1. Further characterization of these more transducible cells and identification of the cellular factors responsible may enhance transduction while maintaining the pluripotentiality of the PHSC.

Cell Cycle↗
PubMed1994

The adenovirus E1A protein overrides the requirement for cellular ras in initiating DNA synthesis.

The adenovirus E1A protein can induce cellular DNA synthesis in growth-arrested cells by interacting with the cellular protein p300 or pRb. In addition, serum- and growth factor-dependent cells require ras activity to initiate DNA synthesis and recently we have shown that Balb/c 3T3 cells can be blocked in either early or late G1 following microinjection of an anti-ras antibody. In this study, the E1A 243 amino acid protein is shown through microinjection not only to shorten the G0 to S phase interval but, what is more important, to override the inhibitory effects exerted by the anti-ras antibody in either early or late G1. Specifically, whether E1A is co-injected with anti-ras into quiescent cells or injected 18 h following a separate injection of anti-ras after serum stimulation, it efficiently induces cellular DNA synthesis in cells that would otherwise be blocked in G0/G1. Moreover, injection of a mutant form of E1A that can no longer associate with p300 is just as efficient as wild-type E1A in stimulating DNA synthesis in cells whose ras activity has been neutralized by anti-ras. The results presented here show that E1A is capable of overriding the requirement of cellular ras activity in promoting the entry of cells into S phase. Moreover, the results suggest the possibility that pRb and/or pRb-related proteins may function in a ras-dependent pathway that enables E1A to achieve this activity.

3T3 Cells↗
PubMed1994

Isolation of a mammalian cell cycle ts mutant, tsJT663, defective in preparation for passing restriction point.

A cell cycle ts mutant, tsJT663, isolated from Fischer rat cell line 3Y1, was characterized. The execution point of tsJT663 was approximately 2 h before S phase, starting either from a G0 (quiescent) state or from the previous cell cycle. Progression of the cell cycle other than from G0/G1 to S phase was temperature insensitive. The restriction (R) point was also estimated as about 2 h before S phase. When tsJT663 cells at log phase were transferred to 40 degrees C, the cell number increased and then decreased within 2 days and thereafter the cells were growth arrested stably for more than 4 weeks. When the serum-stimulated cells were cultured at 34 degrees C for 6 h at 40 degrees C for various period of time and then at 34 degrees C up to the end of the experiments at 30 h, the frequency of cells entering S phase declined depending upon the duration of culture at 40 degrees C. Assuming that the decline of the frequency of tsJT663 cells capable of entering S phase corresponded to the decline of a putative material required for cells to pass the R point toward S phase, the half-life of this material at 40 degrees C was 1 h. These results are consistent with a tentative conclusion that tsJT663 is a cell cycle ts mutant which does not prepare at nonpermissive temperature for passing the restriction point toward S phase.

Animals↗