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Results for “Resting Phase, Cell Cycle”

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PubMed2001

Prosaposin treatment induces PC12 entry in the S phase of the cell cycle and prevents apoptosis: activation of ERKs and sphingosine kinase.

We report that prosaposin treatment induced extracellular signal-regulated kinases (ERKs) and sphingosine kinase activity, increased DNA synthesis, and prevented cell apoptosis. Prosaposin treatment induced pheochromocytoma cells (PC12) to enter the S phase of the cell cycle; this effect was inhibited by the MEK inhibitor PD98059, indicating that prosaposin-induced ERK phosphorylation is required for stimulation of DNA synthesis. The prosaposin effect was also inhibited by pertussis toxin, indicating that the prosaposin receptor is a G-protein-coupled receptor. Prosaposin rescued PC12 cells from apoptosis induced by staurosporine or ceramide. Sphingosine kinase activity was increased by prosaposin treatment. We propose that this effect is a mechanism underlying the proliferative and anti-apoptotic functions of prosaposin. Prosaposin appears to be a key regulatory factor in the ceramide-S-1-P rheostat, which regulates cell fate.

Adrenal Gland Neoplasms↗

PubMed2005

Changing chromatin dynamics and nuclear organization during differentiation in Drosophila larval tissue.

Global changes in gene expression and exit from the cell cycle underlie differentiation. Therefore, understanding chromatin behavior in differentiating nuclei and late G1 is key to understanding this developmental event. A nuclear event that has been shown to specifically occur in late G1 is the association of two heterochromatic blocks in Drosophila. The brown(Dominant) (bw(D)) chromosome of Drosophila melanogaster contains a large block of heterochromatin near the end of 2R. This distal block associates with centric heterochromatin (2Rh), but not until at least 5 hours into G1. We used the bw(D) allele as a model for nuclear organization to determine whether its association with the heterochromatic compartment of the second chromosomes (2Rh) strictly requires differentiation or if this change is a stochastic event, its occurrence being proportional to time spent in G1/G0 phase of the cell cycle. Fluorescence in situ hybridization on eye imaginal discs showed increased association between the bw locus and 2Rh in differentiated cells. Interestingly, an increase in the number of nuclei showing bw(D)-2Rh association in the brains of developmentally delayed larvae that were compromised for differentiation was also observed. Live fluorescence imaging showed that the kinetics of chromatin movement remains unchanged in the developmentally arrested nuclei. These observations suggest that nuclear reorganization is not directly controlled by specific inductive signals during differentiation and that this nuclear reorganization can happen in a cell, regardless of differentiation state, that is arrested in the appropriate cell cycle stage. However, we did see changes that appear to be more directly correlated with differentiation. Dynamic imaging in eye imaginal discs showed that the movement of chromatin is more constrained in differentiated cells, implying that confinement of loci to a smaller nuclear space may help to maintain the changed organization and the transcription profile that accompanies differentiation.

Alleles↗

PubMed2005

IL-2 and autocrine IL-4 drive the in vivo development of antigen-specific Th2 T cells elicited by nematode parasites.

The intestinal nematode parasite, Nippostrongylus brasiliensis, triggers potent type 2 immunity. Using OVA peptide as a model Ag, we have examined the adjuvant effects of this parasite on the in vivo development of Ag-specific Th2 cells from naive DO11.10 T cells. Our findings show that Th2 cells can develop from transferred naive OVA-specific DO11.10 T cells in recipient IL-4-/- mice inoculated with N. brasiliensis plus OVA. However, autocrine IL-4 is required for in situ Th2 cell differentiation since transferred IL-4Ralpha-deficient DO11.10 T cells showed greatly reduced Th2 cell development in inoculated IL-4-/- recipient mice. Surprisingly, we also found that IL-2 blockade promoted B7-dependent T cell cycling, but inhibited the development of OVA-specific Th2 cells. Furthermore, the effects of IL-2 occurred independently of CD25+ T regulatory cells. These studies establish a previously unrecognized requirement for autocrine IL-4 and IL-2 in Th2 responses elicited by nematode parasites.

Amino Acid Sequence↗

PubMed1999

1,25-Dihydroxycholecalciferol (1,25-D3) inhibits the growth of squamous cell carcinoma and down-modulates p21(Waf1/Cip1) in vitro and in vivo.

1,25-Dihydroxycholecalciferol (1,25-D3) has significant antitumor effects in the murine squamous cell carcinoma (SCC) tumor model in vitro and in vivo. We investigated the basis for this antiproliferative activity and found that, in vitro, 1,25-D3 administration is associated with altered expression of cell cycle regulatory proteins, treatment results in retinoblastoma dephosphorylation, decreased expression of p21(Waf1/Cip1) (p21) mRNA and protein, and increased expression of p27Kip1 (p27) mRNA and protein. Dexamethasone, which acts synergistically with 1,25-D3 to inhibit SCC proliferation, enhanced 1,25-D3-induced down-modulation of p21 without affecting the ability of 1,25-D3 to increase p27 expression. 1,25-D3 did not induce cleavage of poly(ADP-ribose) polymerase. These in vitro data suggest that 1,25-D3 exerts antitumor activity in SCC by perturbing cell cycle progression rather than by inducing apoptosis. In vivo, a 1,25-D3 treatment regimen that results in a decrease in SCC tumor volume is associated with a statistically significant decrease in intratumoral p21 expression. p21 expression is not changed in tumors isolated from control animals or animals treated with a nontherapeutic dose of 1,25-D3. Intratumoral p27 levels were not modulated by 1,25-D3 treatment. Thus, both in vitro and in vivo, 1,25-D3-mediated growth inhibition is associated with p21 down-modulation.

Animals↗

PubMed2003

Growth inhibitory and apoptotic effects of inositol hexaphosphate in transgenic adenocarcinoma of mouse prostate (TRAMP-C1) cells.

Inositol hexaphosphate (IP6), a dietary agent, has been extensively studied for its cancer chemopreventive and anti-cancer efficacy in several different animal models, but not in prostate cancer (PCA) possibly because the known PCA models are both expensive and highly time-consuming. One such PCA model is transgenic adenocarcinoma of mouse prostate (TRAMP), which reproduces the spectrum of benign latent, aggressive and metastatic forms of human PCA. In this study, we investigated the anti-cancer effects of IP6 in TRAMP-derived established TRAMP-C1 cell line. IP6 (0.5-4.0 mM) treatment of cells for 24-72 h resulted in 17-76% cell growth inhibition and 6-35% cell death, in a dose- and time-dependent manner. In the studies assessing whether cell growth inhibition by IP6 is associated with an alteration in cell cycle progression, IP6 treatment resulted in up to 92% cells in G0-G1 phase as compared to controls. In other studies assessing its apoptotic efficacy, IP6 induced a moderate to strong (up to 14-fold over control) apoptotic cell death. In additional studies, pretreatment of cells with all caspases inhibitor for 2 h followed by 2 mM IP6 for 48 h resulted in approximately 50% reversal in IP6-induced apoptosis suggesting a partial involvement of caspases activation in IP6-caused apoptosis. Furthermore, IP6 showed significant induction (6-fold) in caspase-3 activity compared to control suggesting the involvement of caspases activation in IP6-induced apoptosis. However, pretreatment of cells with all caspase inhibitor, which partially reversed the IP6-induced apoptosis, completely inhibited IP6-induced caspase-3 activation, providing convincing evidence of both caspase-dependent and -independent mechanisms in IP6-induced apoptotic death of TRAMP-C1 cells. Together, these results warrant further mechanistic and in vivo efficacy studies with IP6 in TRAMP and other pre-clinical models of PCA.

Adenocarcinoma↗

PubMed1996

Terminally differentiated skeletal myotubes are not confined to G0 but can enter G1 upon growth factor stimulation.

Terminally differentiated cells are specialized cells unable to proliferate that constitute most of the mammalian body. Despite their abundance, little information exists on the characteristics of cell cycle control in these cells and the molecular mechanisms that prevent their proliferation. They are generally believed to be irreversibly restricted to the G0 state. In this report, we define some features of a paradigmatic terminally differentiated system, the skeletal muscle, by studying its responses to various mitogenic stimuli. We show that forced expression of a number of cell cycle-regulatory genes, including erbB-2, v-ras, v-myc, B-myb, ld-1, and E2F-1, alone or in combinations, cannot induce terminally differentiated skeletal muscle cells (myotubes) to synthesize DNA. However, serum-stimulated myotubes display a typical immediate-early response, including the up-regulation of c-fos, c-jun, c-myc, and ld-1. They also elevate the expression of cyclin D1 after 4 hours of serum treatment. All these events take place in myotubes in a way that is indistinguishable from that of quiescent, undifferentiated myoblasts reactivated by serum. Moreover, pretreatment with serum shortens the time required by E1A to induce DNA synthesis, confirming that myotubes can partially traverse G1. Serum growth factors do not activate late-G1 genes in myotubes, suggesting that the block that prevents terminally differentiated cells from proliferating acts in mid-G1. Our results show that terminally differentiated cells are not confined to G0 but can partially reenter G1 in response to growth factors; they contribute to a much-needed definition of terminal differentiation. The important differences in the control of the cell cycle between terminally differentiated and senescent cells are discussed.

Adenovirus E1A Proteins↗

PubMed1995

Gas1-induced growth suppression requires a transactivation-independent p53 function.

In normal cells, induction of quiescence is accompanied by the increased expression of growth arrest-specific genes (gas). One of them, gas1, is regulated at the transcriptional level and codes for a membrane-associated protein (Gas1) which is down regulated during the G0-to-S phase transition in serum-stimulated cells. Gas1 is not expressed in growing or transformed cells, and when overexpressed in normal fibroblasts, it blocks the G0-to-S phase transition. Moreover, Gas1 blocks cell proliferation in several transformed cells with the exception of simian virus 40- or adenovirus-transformed cell lines. In this paper, we demonstrate that overexpression of Gas1 blocks cell proliferation in a p53-dependent manner and that the N-terminal domain-dependent transactivating function of p53 is dispensable for Gas1-induced growth arrest. These data therefore indicate that the other intrinsic transactivation-independent functions of p53, possibly related to regulation of apoptosis, should be involved in mediating Gas1-induced growth arrest.

3T3 Cells↗

PubMed1997

Bovine herpesvirus 1-induced apoptosis occurs at the G0/G1 phase of the cell cycle.

We have previously shown that bovine herpesvirus 1 (BHV-1), even when inactivated, induces apoptotic cell death in mitogen-stimulated bovine peripheral blood mononuclear cells (PBMCs) (Hanon et al., 1996, J. Virol. 70, 4116-4120). In order to gain insight into this process, we have investigated the cell cycle phase at which BHV-1 induces apoptosis in PBMCs. Our results show that the percentage of cells that progress through the S phase was always lower in BHV-1-infected PBMCs than in control cells. This effect was not due to a defective activation of mitogen-stimulated PBMCs since BHV-1 only slightly affected the percentage of cells expressing BoCD25, a well-known lymphocyte activation marker. Furthermore, mimosine and cyclosporine A, two chemicals that inhibit entry into the S phase of the cell cycle by different pathways, did not affect the ability of BHV-1 to induce apoptosis. BHV-1-induced apoptosis also occurred in unstimulated PBMCs and interestingly, this was associated with the expression of c-myc and BoCD25 proteins both of which are related to cell cycle progression. All together, these data provide evidence demonstrating that BHV-1-induced apoptosis occurs at the G0/G1 phase of the cell cycle.

Animals↗

PubMed1993

The role of a PDGF-activated nonselective cation channel in the proliferative response.

Murine fibroblasts have a 28 pS calcium- and voltage-insensitive NSC that becomes quiescent at G0 arrest and is rapidly and specifically activated by PDGF. Activation is produced by the discrete loss of long channel closures. The NSC can be rapidly and reversibly blocked with the NSAID flufenamic acid, through a prostaglandin-independent mechanism. The cell cycle (not viability) is blocked concomitantly with NSC block. A somatic cell mutant with altered NSC conductance has been isolated and used to clone the genomic locus of the channel. The mutant growth phenotype adds further support to the participation of NSC conductance in cell cycle control.

Animals↗

PubMed1999

Cell cycle arrest during measles virus infection: a G0-like block leads to suppression of retinoblastoma protein expression.

One of the major mechanisms by which measles virus (MV) infection causes disease and death is suppression of the immune response. The nonresponsiveness of MV-infected human lymphocytes to mitogens and a partial block in the G0/G1 phase of the cell cycle observed in vitro is thought to reflect in vivo immunosuppression. In order to molecularly dissect MV-induced immunosuppression, we analyzed expression of surface activation markers and cell cycle-regulatory proteins in MV-infected human T lymphocytes. MV Edmonston (MV-Ed) could induce and maintain a high level of the early activation marker CD69 in the absence of proliferation. Expression of cyclins D3 and E, which positively control entry into S phase, was also significantly decreased. Analysis of inhibitors of progression into S phase showed that a high level of p27 was maintained in the G0/G1-blocked subpopulation of MV-Ed-infected cells compared to the proliferating MV-infected cells. Furthermore, cell cycle-related upregulation of retinoblastoma (Rb) protein synthesis did not occur in the MV-Ed-infected lymphocytes. Acridine orange staining, which distinguishes cells in G0 from cells in G1, showed that RNA levels were not upregulated following activation, which is consistent with cells remaining in a G0 state. Although expression of surface activation markers indicated entry into the cycle, intracellular Rb and RNA levels suggested a quiescent state. These results indicate that MV can uncouple activation of T lymphocytes from transition of G0 to G1.

Antigens, CD↗

PubMed1995

A novel Ca2+ entry mechanism is turned on during growth arrest induced by Ca2+ pool depletion.

Ca2+ pool depletion with Ca2+ pump blockers induces growth arrest of rapidly dividing DDT1MF-2 smooth muscle cells and causes cells to enter a stable, quiescent G0-like growth state (Short, A.D., Bian, J., Ghosh, T.K., Waldron, R.T., Rybak, S.L., and Gill, D.L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4986-4990). Here we reveal that induction of this quiescent growth state with the Ca2+ pump blocker, thapsigargin, is correlated with the appearance of a novel caffeine-activated Ca2+ influx mechanism. Ca2+ influx through this mechanism is clearly distinct from and additive with Ca2+ entry through store-operated channels (SOCs). Whereas SOC-mediated entry is activated seconds after Ca2+ pool release, caffeine-sensitive influx requires at least 30 min of pool emptying. Although activated in the 1-10 mM caffeine range, this mechanism has clearly distinct methylxanthine specificity from ryanodine receptors and is not modified by ryanodine. It is also unaffected by the Ca2+ channel blockers SKF96365 or verapamil and is independent of modifiers of cyclic nucleotide levels. Growth arrest by thapsigargin-induced Ca2+ pool depletion can be reversed by treatment with 20% serum (Waldron, R.T., Short, A.D., Meadows, J.J., Ghosh, T.K., and Gill, D.L. (1994) J. Biol. Chem. 269, 11927-11933). The serum-induced return of functional Ca2+ pools and reentry of cells into the cell cycle correlates exactly with the disappearance of the caffeine-sensitive Ca2+ influx mechanism. Therefore, appearance and function of this novel Ca2+ entry mechanism are closely tied to Ca2+ pool function and cell growth state and may provide an important means for modifying exit from or entry into the cell cycle.

Animals↗

PubMed2005

Tetrazolium violet induces G0/G1 arrest and apoptosis in brain tumor cells.

Tetrazolium violet (TV), a potent anticancer agent, has been shown to induce cell growth-inhibition in tumor cells. However, the related mechanism has not been revealed yet. In this report we assessed the influence of TV on cell growth and cell cycle in brain tumor cells. Treatment of C6 tumor cells with TV (5-15 microM for 24-72 h) resulted in a growth inhibition in a dose and time-dependent manner and G0/G1 phase arrest, determined by flow cytometry analysis. These effects were accompanied by apoptosis other than necrosis, evidenced by nuclear condensation, terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay and trypan blue exclusion assay plus lactate dehydrogenase (LDH) release assay. Treatment of cells with TV at 15 microM for 24 h resulted in an increase in the activity of caspase-3, evidenced by colorimetric assay, and a dramatic up-regulation of p53, accompanied with a significant increase of Bax/Bcl-2 ratio, as evidenced by immunofluorescence assay. These results suggest that TV induces growth inhibition of C6 cells through p53-midiated apoptotic pathway and G0/G1 checkpoint mechanism. Although detailed mechanisms remain to be explored, selective blockage of tumor cells in G0/G1 phase accompanied by p53-associated apoptosis makes tetrazolium violet a promising anticancer agent, meriting further investigations.

Antineoplastic Agents↗

PubMed2005

Expression of progesterone receptor B is associated with G0/G1 arrest of the cell cycle and growth inhibition in NIH3T3 cells.

Previously, we found a significant reduction of progesterone receptor B (PR-B) expression levels in the Ras-mediated NIH3T3 cell transformation, and re-expression of exogenous PR-B eliminated the tumorigenic potential. We hypothesized that this reduction is of biological significance in cell transformation. In the present study, we determined the correlation between PR-B expression and cell cycle progression. In synchronized NIH3T3 cells, we found an increase in PR-B protein and p27 CDK inhibitor levels in the G0/G1 phase and a reduction due to redistribution in the S and G2/M phases. The MEK inhibitor or cAMP stimulation arrested NIH3T3 cells in the G0/G1 phase of the cell cycle. The expression of PR-B and p27 CDK inhibitors was up-regulated by treatment with both the MEK inhibitor and cAMP. Treatment of synchronized cells with a PKA inhibitor in the presence of 1% calf serum resulted in a significant reduction in both PR-B and p27 levels. The decrease in the PR-B levels caused by anti-sense oligomers or siRNA corresponded to the reduction in p27 levels. PR-B overexpression by adenovirus infection induced p27 and suppressed cell growth. Finally, we showed that PR-B modulation involved in the regulation of NIH3T3 cell proliferation was independent of nuclear estrogen receptor (ER) activity but dependent on non-genomic ER activity.

Adenoviridae↗

PubMed1998

A new model of cell cycle-regulated transcription: repression of the cyclin A promoter by CDF-1 and anti-repression by E2F.

Cell cycle regulation of the cyclin A gene is determined by a bipartite repressor binding site in the region of the basal promoter, termed CDE-CHR, which also controls the expression of cell cycle genes upregulated in S or G2 (such as cdc25C). The CDE-CHR in the cyclin A promoter is recognized by both E2F complexes and CDF-1, but the contribution of each of these factors in cell cycle regulation is unknown. In the present study, we have introduced mutations into the cyclin A promoter which lead to either a loss or enhancement of E2F binding, while having only marginal effects on the interaction with CDF-1. Unlike mutants deficient for CDF-1 binding, promoter variants lacking E2F binding showed an unchanged repression in G0, thus identifying CDF-1 as the principal repressor of the cyclin A gene. The same mutants did show, however, a delayed derepression while a mutation leading to increased E2F binding resulted in premature up-regulation. These findings clearly suggest that E2F contributes to the correct timing of cyclin A transcription, presumably by acting as an anti-repressor. In agreement with this conclusion, we find that the cyclin A promoter only poorly interacts with E2F-4, which is the major E2F family member in G0 cells, while a clear binding is seen with E2F-1 and -3, which are up-regulated in late G1.

3T3 Cells↗

PubMed2000

Differential mRNA expression of the human DNA methyltransferases (DNMTs) 1, 3a and 3b during the G(0)/G(1) to S phase transition in normal and tumor cells.

DNA methylation is essential for mammalian development, X-chromosome inactivation, and imprinting yet aberrant methylation patterns are one of the most common features of transformed cells. One of the proposed causes for these defects in the methylation machinery is overexpression of one or more of the three known catalytically active DNA methyltransferases (DNMTs) 1, 3a and 3b, yet there are clearly examples in which overexpression is minimal or non-existent but global methylation anomalies persist. An alternative mechanism which could give rise to global methylation errors is the improper expression of one or more of the DNMTs during the cell cycle. To begin to study the latter possibility we examined the expression of the mRNAs for DNMT1, 3a and 3b during the cell cycle of normal and transformed cells. We found that DNMT1 and 3b levels were significantly downregulated in G(0)/G(1)while DNMT3a mRNA levels were less sensitive to cell cycle alterations and were maintained at a slightly higher level in tumor lines compared to normal cell strains. Enzymatic activity assays revealed a similar decrease in the overall methylation capacity of the cells during G(0)/G(1)arrest and again revealed that a tumor cell line maintained a higher methylation capacity during arrest than a normal cell strain. These results reveal a new level of control exerted over the cellular DNA methylation machinery, the loss of which provides an alternative mechanism for the genesis of the aberrant methylation patterns observed in tumor cells.

Base Sequence↗

PubMed1993

Cell cycle-specific association of E2F with the p130 E1A-binding protein.

Association of the E2F transcription factor with the pRb and p107 proteins appears to regulate the activity of E2F and, in turn, affect cell cycle progression. We found, however, that pRb and p107 are only minor E2F-associated proteins in G0/G1 mouse fibroblasts, and we sought to identify the major E2F partner protein in these cells. Because the adenovirus E1A oncoprotein seemed able to bind to the G0 E2F partner, we enriched for proteins that associated both with an E2F-binding site DNA column and with E1A. The major species in G0 and early G1 fibroblasts detected with this approach had properties identical to the pRb- and p107-related p130 protein. In serum-stimulated cells, p107 replaced p130 as the major E2F-associated protein near the G1/S border, concomitant with an increase in p107 protein levels. p130-E2F complexes resembled p107-E2F complexes in their ability to bind to cyclin-cdk kinases, and they appeared to be associated with the cyclin E-cdk2 kinase in late G1 cells. These observations indicate that E2F transcription factors are regulated by a succession of partner proteins with which they associate during defined stages of the cell cycle.

3T3 Cells↗

PubMed1991

A cell cycle ts mutant, tsJT16, is defective in p70 synthesis through protein kinase C-dependent and -independent pathways.

tsJT16 is a G0/G1 ts mutant from the Fischer rat fibroblast line. It has a ts defect in a function operating early after growth stimulation with fetal bovine serum (FBS). A primarily induced gene product, p70, was not synthesized at 40 degrees C after stimulation with serum, while c-fos and c-myc mRNAs accumulated under the same condition. This paper reports that p70 was synthesized following stimulation of G0-arrested cells with platelet-derived growth factor, epidermal growth factor (EGF), and 12-0-tetradecanoylphorbol-13-acetate (TPA) at 34 degrees C, but not at 40 degrees C. However, it was synthesized at both temperatures after addition of A23187. In protein kinase C-deprived cells, peptide growth factors and A23187 induced p70 at 34 degrees C, whereas TPA did not. Fibroblast growth factor and insulin did not induce p70. Induction of c-fos and c-myc occurred at both temperatures after the stimulation with FBS, TPA or A23187. These results indicated that the defect in tsJT16 to induce p70 is likely to be located at the common downstream of protein kinase C-dependent and -independent pathways, but is independent from the pathway of calcium mobilization.

Animals↗

PubMed2003

Are chromosomal instabilities induced by exposure of cultured normal human cells to low- or high-LET radiation?

Radiation-induced genomic instability has been proposed as a very early, if not an initiating, step in radiation carcinogenesis. Numerous studies have established the occurrence of radiation-induced chromosomal instability in various cells of both human and rodent origin. In many of these studies, however, the cells were not "normal" initially, and in many cases they involved tumor-derived cell lines. The phenomenon clearly would be of even greater interest if it were shown to occur generally in cells that are normal at the outset, rather than cells that may have been "selected" because of a pre-existing susceptibility to induced instability. As a test of the generality of the phenomenon, we studied low-passage normal diploid human fibroblasts (AG1521A) to determine whether they are susceptible to the induction of chromosomal instability in the progeny of surviving cells after exposure in G(0) to low- and high-LET radiation. Cytogenetic assays for instability were performed on both mixed populations of cells and clones of cells surviving exposure. We found no evidence for the induction of such instability as a result of radiation exposure, though we observed a senescence-related chromosomal instability in the progeny of both irradiated and unirradiated cell populations.

Cell Cycle↗