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Results for “Resting Phase, Cell Cycle”

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PubMed2005

The antiangiogenic factor, 16-kDa human prolactin, induces endothelial cell cycle arrest by acting at both the G0-G1 and the G2-M phases.

The 16-kDa N-terminal fragment of human prolactin (16K hPRL) is a potent antiangiogenic factor that has been shown to prevent tumor growth in a xenograph mouse model. In this paper we first demonstrate that 16K hPRL inhibits serum-induced DNA synthesis in adult bovine aortic endothelial cells. This inhibition is associated with cell cycle arrest at both the G(0)-G(1) and the G(2)-M phase. Western blot analysis revealed that 16K hPRL strongly decreases levels of cyclin D1 and cyclin B1, but not cyclin E. The effect on cyclin D1 is at least partially transcriptional, because treatment with 16K hPRL both reduces the cyclin D1 mRNA level and down-regulates cyclin D1 promoter activity. This regulation may be due to inhibition of the MAPK pathway, but it is independent of the glycogen synthase kinase-3beta pathway. Lastly, 16K hPRL induces the expression of negative cell cycle regulators, the cyclin-dependent kinase inhibitors p21(cip1) and p27(kip1). In summary, 16K hPRL inhibits serum-induced proliferation of endothelial cells through combined effects on positive and negative regulators of cell cycle progression.

Angiogenesis Inhibitors↗

PubMed2006

A new description of cellular quiescence.

Cellular quiescence, defined as reversible growth/proliferation arrest, is thought to represent a homogenous state induced by diverse anti-mitogenic signals. We used transcriptional profiling to characterize human diploid fibroblasts that exited the cell cycle after exposure to three independent signals--mitogen withdrawal, contact inhibition, and loss of adhesion. We show here that each signal caused regulation of a unique set of genes known to be important for cessation of growth and division. Therefore, contrary to expectation, cells enter different quiescent states that are determined by the initiating signal. However, underlying this diversity we discovered a set of genes whose specific expression in non-dividing cells was signal-independent, and therefore representative of quiescence per se, rather than the signal that induced it. This fibroblast "quiescence program" contained genes that enforced the non-dividing state, and ensured the reversibility of the cell cycle arrest. We further demonstrate that one mechanism by which the reversibility of quiescence is insured is the suppression of terminal differentiation. Expression of the quiescence program was not simply a downstream consequence of exit from the cell cycle, because key parts, including those involved in suppressing differentiation, were not recapitulated during the cell cycle arrest caused by direct inhibition of cyclin-dependent kinases. These studies form a basis for understanding the normal biology of cellular quiescence.

Cell Adhesion↗

PubMed1992

Repression of a G0-associated 65-kilodalton protein in actively proliferating and SV40-transformed mouse kidney cells.

The pattern of [35S]methionine-labeled proteins from primary cultures of mouse kidney epithelial cells arrested in G0 phase was analyzed by two-dimensional gel electrophoresis and compared with that observed from cultures of actively proliferating and SV40-transformed mouse kidney cells. A major polypeptide (p65) migrating with a molecular mass of 65,000 daltons and a pI of 5.8 was detected in quiescent cultures of cells which had exhausted their finite division potential. Under the experimental conditions used, these cells had lost sensitivity to growth factors and were irreversibly blocked in G0 phase of the cell cycle. In cultures of actively proliferating mouse kidney cells, the expression of p65 was not observed until just prior to arrest. Moreover, proliferating cultures of immortalized mouse kidney cells that had been reactivated from their quiescent state by infection with SV40 did not express p65. Subcellular localization studies suggest that p65 is associated with the crude nuclear fraction. In addition, p65 is glycosylated and binds the lectin concanavalin A. Pulse-chase experiments demonstrated that p65 was short lived with an estimated half life of 10 min. Thus, p65 appears to be a growth-arrest specific gene product whose expression is repressed during the proliferative state of mitotically active mouse kidney cells.

Animals↗

PubMed1995

Repression of platelet-derived growth factor beta-receptor expression by mitogenic growth factors and transforming oncogenes in murine 3T3 fibroblasts.

Platelet-derived growth factor BB (PDGF-BB) is an important extracellular factor for regulating the G0-S phase transition of murine BALB/c-3T3 fibroblasts. We have investigated the expression of the PDGF beta receptor (PDGF beta R) in these cells. We show that the state of growth arrest in G0, resulting from serum deprivation, is associated with increased expression of the PDGF beta R. When the growth-arrested fibroblasts are stimulated to reenter the cell cycle by the mitogenic action of serum or certain specific combinations of growth factors, PDGF beta R mRNA levels and cell surface PDGF-BB-binding sites are markedly downregualted. Oncogene-transformed 3T3 cell lines, which fail to undergo growth arrest following prolonged serum deprivation, express constitutively low levels of the PDGF beta R mRNA and possess greatly reduced numbers of cell surface PDGF receptors, as determined by PDGF-BB binding and Western blotting (immunoblotting). Nuclear runoff assays indicate the mechanism of repression of PDGF beta R expression to be, at least in large part, transcriptional. These data indicate that expression of the PDGF beta R is regulated in a growth state-dependent manner in fibroblasts and suggest that this may provide a means by which cells can modulate their responsiveness to the actions of PDGF.

3T3 Cells↗

PubMed2003

Dictyostelium differentiation-inducing factor-3 activates glycogen synthase kinase-3beta and degrades cyclin D1 in mammalian cells.

In search of chemical substances applicable for the treatment of cancer and other proliferative disorders, we studied the signal transduction of Dictyostelium differentiation-inducing factors (DIFs) in mammalian cells mainly using HeLa cells. Although DIF-1 and DIF-3 both strongly inhibited cell proliferation by inducing G(0)/G(1) arrest, DIF-3 was more effective than DIF-1. DIF-3 suppressed cyclin D1 expression at both mRNA and protein levels, whereas the overexpression of cyclin D1 overrode DIF-3-induced cell cycle arrest. The DIF-3-induced decrease in the amount of cyclin D1 protein preceded the reduction in the level of cyclin D1 mRNA. The decrease in cyclin D1 protein seemed to be caused by accelerated proteolysis, since it was abrogated by N-acetyl-Leu-Leu-norleucinal, a proteasome inhibitor. DIF-3-induced degradation of cyclin D1 was also prevented by treatment with lithium chloride, an inhibitor of glycogen synthase kinase-3beta (GSK-3beta), suggesting that DIF-3 induced cyclin D1 proteolysis through the activation of GSK-3beta. Indeed, DIF-3 dephosphorylated Ser(9) and phosphorylated tyrosine on GSK-3beta, and it stimulated GSK-3beta activity in an in vitro kinase assay. Moreover, DIF-3 was revealed to induce the nuclear translocation of GSK-3beta by immunofluorescent microscopy and immunoblotting of subcellular protein fractions. These results suggested that DIF-3 activates GSK-3beta to accelerate the proteolysis of cyclin D1 and that this mechanism is involved in the DIF-3-induced G(0)/G(1) arrest in mammalian cells.

Animals↗

PubMed2004

Imatinib mesylate inhibits T-cell proliferation in vitro and delayed-type hypersensitivity in vivo.

Imatinib mesylate (STI571, imatinib) inhibited DNA synthesis in primary human T cells stimulated with allogeneic mature dendritic cells or phytohemagglutinin (PHA) but did not induce apoptosis. The values for the concentration that inhibits 50% (IC50) of T-cell proliferation stimulated by dendritic cells and PHA were 3.9 microM and 2.9 microM, respectively, that is, within the concentration range found in patients treated with imatinib mesylate. Interestingly, imatinib mesylate did not inhibit expression of T-cell activation markers CD25 and CD69, although it reduced the levels of activated nuclear factor-kappaB (NF-kappaB) and changed phosphorylation or protein levels of Lck, ERK1/2, retinoblastoma protein, and cyclin D3. When T cells were washed free of imatinib mesylate, they proliferated in response to PHA, demonstrating that inhibition is reversible. Treatment with imatinib mesylate led to accumulation of the cells in G0/G1 phase of the cell cycle. The in vitro observations were confirmed in vivo in a murine model of delayed-type hypersensitivity (DTH). In mice treated with imatinib mesylate, DTH was reduced in comparison to sham-injected controls. However, the number of splenic T cells was not reduced showing that, similarly to in vitro observations, imatinib mesylate inhibited T-cell response, but did not cause apoptosis. These findings indicate that long-term administration of high-dose imatinib mesylate might affect immunity.

Animals↗

PubMed1999

G1 versus G2 cell cycle arrest after adriamycin-induced damage in mouse Swiss3T3 cells.

Cell cycle arrest after different types of DNA damage can occur in either G1 phase or G2 phase of the cell cycle, involving the distinct mechanisms of p53/p21(Cip1/Waf1) induction, and phosphorylation of Cdc2, respectively. Treatment of asynchronously growing Swiss3T3 cells with the chemotherapeutic drug adriamycin induced a predominantly G2 cell cycle arrest. Here we investigate why Swiss3T3 cells were arrested in G2 phase and not in G1 phase after adriamycin-induced damage. We show that adriamycin was capable of inducing a G1 cell cycle arrest, both during the G0-G1 transition and during the G1 phase of the normal cell cycle. In G0 cells, adriamycin induced a prolonged cell cycle arrest. However, adriamycin caused only a transient cell cycle delay when added to cells at later time points during G0-G1 transition or at the G1 phase of normal cell cycle. The G1 arrest correlated with the induction of p53 and p21(Cip1/Waf1), and the exit from the arrest correlated with the decline of their expression. In contrast to the G1 arrest, adriamycin-induced G2 arrest was relatively tight and correlated with the Thr-14/Tyr-15 phosphorylation of cyclin B-Cdc2 complexes. The relative stringency of the G1 versus G2 cell cycle arrest may explain the predominance of G2 arrest after adriamycin treatment in mammalian cells.

3T3 Cells↗

PubMed2003

p53 Is required for 1,25-dihydroxyvitamin D3-induced G0 arrest but is not required for G1 accumulation or apoptosis of LNCaP prostate cancer cells.

1,25-Dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] is an effective agent for inhibiting the growth of prostate cancer cells including LNCaP and PC-3 cell lines. However, the extent of growth inhibition in these cell lines differs because LNCaP cells are much more responsive than PC-3 cells. Previous studies in LNCaP cells have shown that 1,25-(OH)(2)D(3) treatment results in G(0)/G(1) cell cycle accumulation, loss of Ki67 expression, and induction of apoptosis. One difference between the two cell lines is that PC-3 cells lack functional p53, a protein that plays roles both in cell cycle regulation and induction of apoptosis. In this study, the role of p53 in 1,25-(OH)(2)D(3) action was examined using the p53-negative PC-3 cells and a line of LNCaP cells, called LN-56, in which p53 function was shut off using a dominant negative p53 fragment. We found that treatment with 1,25-(OH)(2)D(3) extensively inhibits growth of LN-56 prostate cancer cells lacking p53, but in contrast to the parental LNCaP cells, the LN-56 cells recover rapidly. Moreover, in prostate cancer cells, the synergism between 1,25-(OH)(2)D(3) and 9-cis retinoic acid appears to be dependent on the presence of functional p53; however, 1,25-(OH)(2)D(3)-mediated induction of G(1) cell cycle accumulation and induction of apoptosis is not.

Alitretinoin↗

PubMed2002

Stromal cell-derived factor 1 regulates primitive hematopoiesis by suppressing apoptosis and by promoting G(0)/G(1) transition in CD34(+) cells: evidence for an autocrine/paracrine mechanism.

The stromal cell-derived factor 1 (SDF-1) chemokine has various effects on hematopoietic cell functions. Its role in migration and homing of hematopoietic progenitors is currently well established. Previously it was shown that SDF-1 stimulates myeloid progenitor proliferation in synergy with cytokines. Results of this study indicate that SDF-1 alone promotes survival of purified CD34(+) cells from human unmobilized peripheral blood (PB) by counteracting apoptosis as demonstrated by its capacity to reduce DNA fragmentation, annexin-V(+) cell number, and APO2.7 detection and to modulate bcl-2 homolog protein expression. The study demonstrates that SDF-1, produced by sorted CD34(+)CD38(+) cells and over-released in response to cell damage, exerts an antiapoptotic effect on CD34(+) cells through an autocrine/paracrine regulatory loop. SDF-1 participates in the autonomous survival of circulating CD34(+) cells and its effect required activation of the phosphotidyl inositol 3 kinase (PI3-K)/Akt axis. Cell sorting based on Hoechst/pyroninY fluorescences shows that SDF-1 production is restricted to cycling CD34(+) cells. SDF-1 triggers G(0) quiescent cells in G(1) phase and, in synergy with thrombopoietin or Steel factor, makes CD34(+) cells progress through S+G(2)/M phases of cell cycle. By assessing sorted CD34(+)CD38(-) and CD34(+)CD38(+) in semisolid culture, the study demonstrates that SDF-1 promotes survival of clonogenic progenitors. In conclusion, the results are the first to indicate a role for endogenous SDF-1 in primitive hematopoiesis regulation as a survival and cell cycle priming factor for circulating CD34(+) cells. The proposal is made that SDF-1 may contribute to hematopoiesis homeostasis by participating in the autonomous survival and cycling of progenitors under physiologic conditions and by protecting them from cell aggression in stress situations.

Antigens, CD34↗

PubMed1991

The phosphorylation state of the retinoblastoma (RB) protein in G0/G1 is dependent on growth status.

The product of the retinoblastoma gene (RB) is a nuclear phosphoprotein which is thought to regulate the proliferation of cells. Its phosphorylation state changes with passage through the cell cycle and it has been proposed that RB protein in its hypo-phosphorylated form prevents cells proliferating. We have investigated the phosphorylation state of the RB protein in an actively-dividing human B-lymphoblastoid cell line and after cell cycle arrest caused by alpha-Interferon (alpha-IFN). We show that the phosphorylation state of the RB protein in cells with 2N DNA content depends on whether the cells are actively cycling. Our data is compatible with the proposal that dephosphorylation of the RB protein allows cells to enter a quiescent state. This study sheds light on the molecular mechanisms which may mediate the cytostatic effects of alpha-IFN.

Cell Line↗

PubMed2002

Oligosaccharide-linked acyl carrier protein, a novel transmethylase inhibitor, from porcine liver inhibits cell growth.

We have previously reported on the identification of the endogenous transmethylation inhibitor oligosaccharide-linked acyl carrier protein (O-ACP). In this study, the role of the transmethylation reaction on cell cycle progression was evaluated using various transmethylase inhibitors, including O-ACP. O-ACP significantly inhibited the growth of various cancer cell lines, including NIH3T3, ras-transformed NIH3T3, MDA-MB-231, HT-1376, and AGS. In addition, exposure of ras-transformed NIH3T3 to O-ACP caused cell cycle arrest at the G0/G1 phase, which led to a decrease in cells at the S phase, as determined by flow cytometry. In contrast, transmethylase inhibitors did not affect the expression of p21(WAF1/Cip1), a well known inhibitor of cyclin dependent kinase, indicating that the cell cycle arrest by transmethylase inhibitors might be mediated by a p21(WAF1/Cip1)-independent mechanism. Therefore, O-ACP, a novel transmethylase inhibitor, could be a useful tool for elucidating the novel role of methylation in cell proliferation and cell cycle progression.

3T3 Cells↗

PubMedDate not supplied

[The effect of inhibitors of DNA and protein synthesis on the chromosome aberration yield in a human lymphocyte culture exposed to gamma and neutron irradiation at different stages of the mitotic cycle. The cytogenetic effects in the G0 phase].

The effect of inhibitors of protein synthesis, cycloheximide (CH), and DNA synthesis, hydroxyurea (OU) in combination with 5-fluoro-2-deoxyuridine (OU + FDUR), on the yield of chromosome aberrations in cultured human lymphocytes induced by gamma- and neutron irradiation with doses of 1 and 2 Gy in G0 phase of mitotic cycle has been studied. The inhibitors added immediately after gamma-irradiation have caused reliable increase of aberration rate (CH at both doses and OU + FDUR at a dose of 2 Gy only). Both inhibitors caused no effect being added after 1.5, 3 and 5 hr after gamma-irradiation. The chromosome aberration rate substantially increased at both doses of neutron irradiation for both inhibitors added at all studied time intervals after exposure. Chromosome exchange aberrations have most contributed to the increase of aberration rate.

Blood Proteins↗

PubMed2006

All-trans retinoic acid induces p62DOK1 and p56DOK2 expression which enhances induced differentiation and G0 arrest of HL-60 leukemia cells.

p62(DOK1) (DOK1) and p56(DOK2) (DOK2) are sequence homologs that act as docking proteins downstream of receptor or nonreceptor tyrosine kinases. Originally identified in chronic myelogenous leukemia cells as a highly phosphorylated substrate for the chimeric p210(bcr-abl) protein, DOK1 was suspected to play a role in leukemogenesis. However, p62(DOK1-/-) fibroblast knockout cells were found to have enhanced MAPK signaling and proliferation due to growth factors, suggesting negative regulatory capabilities for DOK1. The role of DOK1 and DOK2 in leukemogeneis thus is enigmatic. The data in this report show that both the DOK1 and the DOK2 adaptor proteins are constitutively expressed in the myelomonoblastic leukemia cell line, HL-60, and that expression of both proteins is induced by the chemotherapeutic differentiation causing agents, all-trans retinoic acid (atRA) and 1,25-dihydroxyvitamin D3 (VD3). Ectopic expression of either protein enhances atRA- or VD3-induced growth arrest, differentiation, and G(0)/G(1) cell cycle arrest and results in increased ERK1/2 phosphorylation. DOK1 and DOK2 are similarly effective in these capabilities. The data provide evidence that DOK1 and DOK2 proteins have a similar role in regulating cell proliferation and differentiation and are positive regulators of the MAPK signaling pathway in this context.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Insulin/IGF-I rescues immortalized brown adipocytes from apoptosis down-regulating Bcl-xS expression, in a PI 3-kinase- and map kinase-dependent manner.

Serum deprivation of immortalized brown adipocyte cell line resulted in growth arrest in G0/G1 phases of the cell cycle and apoptosis, as detected either by DNA laddering or by increase in the percentage of hypodiploid cells. Furthermore, apoptosis is concurrent with a dramatic increase in the expression of the proapoptotic protein Bcl-xS, the expression of Bcl-xL remaining almost undetectable. Insulin/insulin-like growth factor (IGF-I) rescued serum-deprived brown adipocytes from apoptosis, decreasing the number of hypodiploid cells and increasing the number of cells undergoing cell cycle progression throughout S and G2/M phases of the cell cycle. Moreover, insulin down-regulated Bcl-xS expression without inducing the expression of Bcl-xL. Both phosphatidylinositol (PI) 3-kinase and mitogen-activated protein kinase (MAPK) pathways are necessary for insulin/IGF-I full survival effect, since the use of specific inhibitors of PI 3-kinase activity (wortmannin or LY294002, at the dose that inhibits PI 3-kinase activity induced by insulin) or MAPK kinase activity inhibitor (PD098059, at the dose that inhibits insulin-induced phosphorylation of MAPK) totally blocked the antiapoptotic effect induced by insulin/IGF-I, respectively. In conclusion, insulin survival effect on immortalized brown adipocytes is associated with inhibition of the Bcl-xS content without changing Bcl-xL, in a PI 3-kinase- and MAP kinase-dependent manner.

Adipocytes↗

PubMed2005

Transforming growth factor-beta1 inhibits all-trans retinoic acid-induced apoptosis.

The interaction between retinoids and transforming growth factor-beta1 (TGF-beta1) leading to regulation of proliferation, differentiation and apoptosis is not still fully understood. In this study, we demonstrated that a combination treatment with all-trans retinoic acid (ATRA) and TGF-beta1 led to the enhancement of ATRA-induced suppression of cell proliferation, which is accompanied by inhibition of ATRA-induced apoptosis in human leukemia HL-60 cells. This effect was preceded by the arrest of cells in G0/G1 cell cycle phase linked with pRb protein dephosphorylation, continuous accumulation of p21 and transiently increased level of p27, inhibitors of cyclin-dependent kinases. Inhibition of ATRA-induced apoptosis by TGF-beta1 was associated with an increased level of Mcl-1 protein, an anti-apoptotic member of Bcl-2 family, but not with inhibition of mitochondrial membrane depolarization. Levels of other Bcl-2 family proteins (Bcl-2, Bcl-X(L), Bad, Bak, Bax) were unaffected by simultaneous ATRA and TGF-beta1 treatment, when compared to ATRA alone. Upregulation of c-FLIP(L) protein, an inhibitor of apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), correspond with inhibition of ATRA-induced (autocrine TRAIL-mediated) caspase-8 activation and apoptosis. These results suggest that apoptosis inhibition associated with proliferation block could depend on modulation of the TRAIL apoptotic pathway and regulation of the Mcl-1 protein level. In summary, we demonstrate that the balance of processes leading to regulation of proliferation and differentiation of myeloid cells can modulate cell sensitivity to apoptosis-inducing stimuli.

Apoptosis↗

PubMed2003

Molecular targets of 1,25(OH)2D3 in HC11 normal mouse mammary cell line.

Our aim was to determine the molecular targets involved in the antiproliferative effects of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), in a normal murine mammary epithelial cell line, HC11. Among the early response genes analyzed, c-myc, junB, junD, c-jun, c-fos, fosB, fra, as well as max, mad1-4, sin3, only c-jun and fra-2 mRNAs were up-regulated after 1,25(OH)(2)D(3) exposure. Cyclin C was reduced and cyclin A2 and E were slightly enhanced; however, cyclins D1, D3, B1, B2, F, G1, G2, I and H, as well as TGF beta 1, TGF beta 3, T beta RI and T beta RII transcripts were not modulated by 1,25(OH)(2)D(3). Although p27(KIP1) protein content was unchanged, enhancement of p21(WAF1/CIP1) low basal levels in cell extracts and IGFBP-3 abundance on the culture medium was detected after 1,25(OH)(2)D(3) induction. Using differential display analysis, we identified eight down-modulated clones in exposed cells: 26S proteasome non-ATPase subunit Pad1, ubiquitin-conjugating enzyme Ube2i, extracellular proteinase inhibitor Expi or Wdnm1, cytochrome-c oxidase Cox7c, microtubule-associated protein-1 light chain-3 (Map1lc3), nascent-associated complex alpha Naca, transforming acidic coiled-coil Tacc3, stearoyl-CoA desaturase (Scd), keratin 6 alpha, and 1 up-regulated, fork head transcription factor Hfh-1L. Hence, the antiproliferative effect of 1,25(OH)(2)D(3) seems associated to enhancement of c-jun, Fra-2, IGFBP3 and p21(WAF1/CIP1). Decreased Pad1 and Ube2i might account for increased stability of cell cycle inhibitory proteins while reduced Wdnm1, Tacc3 and Scd might be secondary to accumulation of cells in G0/G1 phase.

Animals↗

PubMed2005

A novel platinum compound inhibits constitutive Stat3 signaling and induces cell cycle arrest and apoptosis of malignant cells.

Previous studies have established constitutive activation of Stat3 protein as one of the molecular changes required for tumorigenesis. To develop novel therapeutics for tumors harboring constitutively active Stat3, compounds from the NCI 2000 diversity set were evaluated for inhibition of Stat3 DNA-binding activity in vitro. Of these, a novel platinum (IV) compound, IS3 295, interacted with Stat3 and inhibited its binding to specific DNA-response elements. Further analysis suggested noncompetitive-type kinetics for the inhibition of Stat3 binding to DNA. In human and mouse tumor cell lines with constitutively active Stat3, IS3 295 selectively attenuated Stat3 signaling, thereby inducing cell growth arrest at G0/G1 phase and apoptosis. Moreover, in transformed cells, IS3 295 repressed expression of cyclin D1 and bcl-xL, two of the known Stat3-regulated genes that are overexpressed in malignant cells, suggesting that IS3 295 mediates anti-tumor cell activity in part by blocking Stat3-mediated sub-version of cell growth and apoptotic signals. Together, our findings provide evidence for the inhibition of Stat3 activity and biological functions by IS3 295 through interaction with Stat3 protein. This study represents a significant advance in small molecule-based approaches to target Stat3 and suggests potential new applications for platinum (IV) complexes as modulators of the Stat3 pathway for cancer therapy.

Agar↗

PubMed2004

Nucleolar localization of the human telomeric repeat binding factor 2 (TRF2).

The telomeric repeat binding factor 2 (TRF2) specifically recognizes TTAGGG tandem repeats at chromosomal ends. Unexpectedly immunofluorescence studies revealed a prominent nucleolar localization of TRF2 in human cells, which appeared as discrete dots with sizes similar to those present in the nucleoplasm. The TRF2 dots did not overlap with dots stemming from the upstream binding factor (UBF) or the B23 protein. After treatment with a low concentration of actinomycin D (0.05 microg/ml), TRF2 remained in the nucleolus, although this condition selectively inhibited RNA polymerase I and led to a relocalization of UBF and B23. TRF2 was prominent in the nucleolus at G0 and S but seemed to diffuse out of the nucleolus in G2 phase. During mitosis TRF2 dispersed from the condensed chromosomes and returned to the nucleolus at cytokinesis. Treatment with low doses of actinomycin D delayed the release of TRF2 from the nucleolus as cells progressed from G2 phase into mitosis. With actinomycin D present TRF2 was detected in discrete foci adjacent to UBF in prophase, while in metaphase a complete overlap between TRF2 and UBF was observed. TRF2 was present in DNase-insensitive complexes of nucleolar extracts, whereas DNA degradation disrupted the protein-DNA complexes consisting of Ku antigen and B23. Following treatment with actinomycin D some of the mitotic cells displayed chromosome end-to-end fusions. This could be correlated to the actinomycin D-suppressed relocalization of TRF2 from the nucleolus to the telomeres during mitosis. These results support the view that the nucleolus may sequester TRF2 and thereby influences its telomeric functions.

Animals↗