Mimosine reversibly arrests cell cycle progression at the G1-S phase border

It has previously been demonstrated that the compound mimosine inhibits cell cycle traverse in late G1 phase prior to the onset of DNA synthesis (Hoffman BD, Hanauske-Abel HM, Flint A, Lalande M: Cytometry 12:26-32, 1991; Lalande M: Exp Cell Res 186:332-339, 1990). These results were obtained by using flow cytometric analysis of DNA content to compare the effects of mimosine on cell cycle traverse with those of aphidicolin, an inhibitor of DNA polymerase alpha activity. We have now measured the incorporation of bromodeoxyuridine into lymphoblastoid cells by flow cytometry to determine precisely where the two inhibitors act relative to the initiation of DNA synthesis. It is demonstrated here that mimosine arrests cell cycle progression at the G1-S phase border. The onset of DNA replication occurs within 15 min of releasing the cells from the mimosine block. In contrast, treatment with aphidicolin results in the accumulation of cells in early S phase. These results indicate that mimosine is a suitable compound for affecting the synchronous release of cells from G1 into S phase and for analyzing the biochemical events associated with this cell cycle phase transition.     

A reversible arrest point in the late G1 phase of the mammalian cell cycle

The effects of two different cell cycle inhibitors on the proliferation of human lymphoblastoid cells have been analyzed by flow cytometric techniques. Mimosine, a plant amino acid, reversibly blocks the cell cycle at a point which occurs roughly 2 h before the arrest mediated by aphidicolin, an inhibitor of DNA polymerase alpha activity, which defines the G1/S phase boundary. The levels of thymidine kinase mRNA, which increase at the onset of S phase, are higher in cells blocked with aphidicolin than in cells treated with mimosine whereas the opposite results are obtained in the case of p53 mRNA levels, which are known to be maximal in the late G1 phase. These results indicate that mimosine inhibits cell cycle traverse in the late G1 phase prior to the onset of DNA synthesis and identifies a previously undefined reversible cell cycle arrest point.     

Synthesis of nuclear lamins in BHK-21 cells synchronized with aphidicolin

Lamins A, B and C are the major proteins of mammalian nuclear lamina and have been well studied in BHK-21 cells. By synchronizing BHK-21 cells with aphidicolin, a potent inhibitor of DNA alpha-polymerase, we were able to detect a differential pattern of synthesis for nuclear lamins during the cell cycle. Lamin B starts to be synthesized only in S phase up to mitosis while synthesis of lamins A and C remain stable throughout the cell cycle. The precursor of lamin A see its half-life increase from a reported 63 min in interphase cells to 103 min in G2/M cells.     

Delaying the onset of M phase in NIH 3T3 cells blocked in early S phase occurs via accumulating cyclin B1 and tyrosine-phosphorylated p34cdc2 in the nucleus

An affinity-purified antibody (anti-Cdc2C) raised against the carboxy terminal sequence LDNQIKKM of p34^cdc2 uncovered in NIH 3T3 cells a protein subpopulation, the location and the level of accumulation of which evolve during progression through the cell cycle: it first emerges inside the nucleus in late G1/early S phase and continues to build up principally in this location throughout S phase; a cytoplasmic expression then becomes apparent near the end of S phase, develops during G2 and sometimes prevails over the nuclear expression; it finally relocates to the nucleus in early prophase. We propose that a major part of this subpopulation would represent p34^cdc2 molecules existing inside a complex with cyclin B1. NIH 3T3 cells arrested in early S phase with aphidicolin do not commit prematurely to mitosis which indicates that the regulatory pathway involved in preserving the temporal order of S and M phases is functioning in these conditions. Conjugated Western blot analysis and immunofluorescence microscopy showed that cyclin A, cyclin B1 and tyrosine-phosphorylated p34^cdc2 continue to build up predominantly in the nucleus of the arrested cells. After release from the block, the cells rapidly reenter S and G2 phases and, concomitantly, cyclin B1 and tyrosine-phosphorylated p34^cdc2 relocate to the cytoplasm before redistributing again in the nucleus in early prophase. These data would suggest that delaying the onset of M phase in NIH 3T3 cells in which the rate of DNA replication is reduced, is first ensured by a mechanism that prevents the cytoplasmic relocation of inactive p34^cdc2/cyclin B1 complexes continually forming in the nucleus once the G1 period of mitotic cyclin instability is over.     

Cell cycle dependent gene expressions and activities of protein phosphatases PP1 and PP2A in mouse NIH3T3 fibroblasts.

We determined the mRNA levels and the activities in nuclear and non-nuclear fractions of protein phosphatase type 1 (PP1) and type 2A (PP2A) through the cell cycle in synchronized mouse NIH3T3 fibroblasts. The mRNA level for PP1 alpha was gradually elevated in late G1 phase, began to decrease in M phase, and reached the control level with entering into the next G1 phase. The mRNA level for PP2A was rapidly increased in early G1 phase, kept at the high level, and decreased after S phase. In nuclear fractions of cells, spontaneous activities of both PP1 and PP2A were gradually increased until M phase and rapidly decreased with entering the next G1 phase, while in non-nuclear fraction such dramatic alterations were not observed. Potential activities of PP1 in both fractions revealed by Co(2+)-trypsin treatment showed an oscillaion patterns similar to those of the spontaneous activities. These results strongly suggest that cell cycle dependent gene expressions and activities of PP1 and PP2A play roles in DNA synthesis and mitosis during the cell cycle.     

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

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

PPARgamma1 synthesis and adipogenesis in C3H10T1/2 cells depends on S-phase progression, but does not require mitotic clonal expansion

Adipogenesis is typically stimulated in mouse embryo fibroblast (MEF) lines by a standard hormonal combination of insulin (I), dexamethasone (D), and methylisobutylxanthine (M), administered with a fresh serum renewal. In C3H10T1/2 (10T1/2) cells, peroxisome proliferator-activated receptor gamma1 (PPARgamma1) expression, an early phase key adipogenic regulator, is optimal after 36 h of IDM stimulation. Although previous studies provide evidence that mitotic clonal expansion of 3T3-L1 cells is essential for adipogenesis, we show, here, that 10T1/2 cells do not require mitotic clonal expansion, but depend on cell cycle progression through S-phase to commit to adipocyte differentiation. Exclusion of two major mitogenic stimuli (DM without insulin and fresh serum renewal) from standard IDM protocol removed mitotic clonal expansion, but sustained equivalent PPARgamma1 synthesis and lipogenesis. Different S-phase inhibitors (aphidicolin, hydroxyurea, l-mimosine, and roscovitin) each arrested cells in S-phase, under hormonal stimulation, and completely blocked PPARgamma1 synthesis and lipogenesis. However, G2/M inhibitors effected G2/M accumulation of IDM stimulated cells and prevented mitosis, but fully sustained PPARgamma1 synthesis and lipogenesis. DM stimulation with or without fresh serum renewal elevated DNA synthesis in a proportion of cells (measured by BrdU labeling) and accumulation of cell cycle progression in G2/M-phase without complete mitosis. By contrast, standard IDM treatments with fresh serum renewal caused elevated DNA synthesis and mitotic clonal expansion while achieved equivalent level of adipogenesis. At most, one-half of the 10T1/2 mixed cell population differentiated to mature adipocytes, even when clonally isolated. PPARgamma was exclusively expressed in the cells that contained lipid droplets. IDM stimulated comparable PPARgamma1 synthesis and lipogenesis in isolated cells at low cell density (LD) culture, but in about half of the cells and with sensitivity to G1/S, but not G2/M inhibitors. Importantly, growth arrest occurred in all differentiating cells, while continuous mitotic clonal expansion occurred in non-differentiating cells. Irrespective of confluence level, 10T1/2 cells differentiate after progression through S-phase, where adipogenic commitment induced by IDM stimulation is a prerequisite for PPARgamma synthesis and subsequent adipocyte differentiation.     

Fibroblast growth regulatory factor inhibits DNA synthesis in BALB/c 3T3 cells by arresting in G1

A cell surface macromolecular component from quiescent BALB/c 3T3 mouse cells (designated fibroblast growth regulatory factor, FGRF) inhibits DNA synthesis and cell division in growing 3T3 cells. Addition of FGRF to synchronized populations of growing 3T3 cells in the late G1 or early S phase did not inhibit DNA synthesis in the immediate S phase. However, a significant inhibition was observed in the S phase of the next round of cell cycle. Cells exposed to the regulatory factor in late S/early G2 or early G1 showed reduced DNA synthesis in the upcoming S phase; the late S/early G2 cells were more sensitive to inhibition than the cells in the G1. Further, the regulatory factor delayed the progression of G0/G1-arrested cells into the next S phase. These results suggest that the physiological effect of FGRF is to arrest cells in early G1, thus preventing their entry into a new round of cell cycle. In contrast to untransformed 3T3 cells, mouse cells transformed by SV40 were not subjected to growth-arrest by the regulatory factor, although the transformed cells contain active FGRF that inhibits DNA synthesis in growing 3T3 cells.     

Requirement for phosphatidylinositol-3 kinase activity during progression through S-phase and entry into mitosis

Phosphatidylinositol-3 kinase (PI3K) proteins are important regulators of cell survival and proliferation. PI3K-dependent signalling regulates cell proliferation by promoting G1- to S-phase progression during the cell cycle. However, a definitive role for PI3K at other times during the cell cycle is less clear. In these studies, we provide evidence that PI3K activity is required during DNA synthesis (S-phase) and G2-phase of the cell cycle. Inhibition of PI3K with LY294002 at the onset of S-phase caused a 4- to 5-h delay in progression through G2/M. LY294002 treatment at the end of S-phase caused an approximate 2-h delay in progression through G2/M, indicating that PI3K activity functions for both S- and G2-phase progression. The expression of constitutively activated Akt partially reversed the inhibitory effects of LY294002 on mitotic entry, which demonstrated that Akt was one PI3K target that was required during G2/M transitions. Inhibition of PI3K resulted in enhanced susceptibility of G2/M synchronized cells to undergo apoptosis in response to DNA damage as compared to asynchronous cells. Thus, similar to its role in promoting cell survival and cell cycle transitions from G1 to S phase, PI3K activity appears to promote entry into mitosis and protect against cell death during S- and G2-phase progression.     

Transient peaks in zinc and metallothionein levels during differentiation of 3T3L1 cells

A novel role for zinc mediated by metallothionein (MT) is found in the process of differentiation of 3T3L1 mouse fibroblasts to adipocytes. Twenty-four hours after the stimulation of differentiation by hormones, the cells enter into a phase of synchronous proliferation. In this phase the cellular contents of zinc and metallothionein rise rapidly to fivefold and threefold levels, respectively. Simultaneously MT is translocated from the cytoplasm to the nucleus. The rise of intracellular zinc is essential for the transition from G0/G1- to S-phase of the cell cycle. Deprivation of zinc with N,N,N', N'-tetrakis[2-pyridyl]ethylenediamine, a membrane-permeable zinc chelator, inhibited hormonal induced proliferation. After the short phase of proliferation a slower stage of actual differentiation to adipocytes begins. The elevated levels of MT and zinc decline quickly to start levels, and a rapid redistribution of MT to the cytoplasm occurs. We propose that the nuclear translocation of MT mediates the transfer of zinc to nuclear factors in the mitogenic process. The redistribution of MT to the cytoplasm and the decrease of the zinc content are postulated to be required for the start of the actual differentiation.     

Arrest of 3T3 cells in G1 phase in suspension culture

3T3 cells do not grow in Methocel suspension culture, while other permanent cell lines do. The viability of 3T3 cells in suspension remains unchanged for at least three days with respect to plating efficiency, vital staining and resumption of normal growth when transferred into monolayer culture. When monolayer 3T3 cells in G1 phase are suspended they remain in G1 phase. Cells already in S phase which are suspended complete ongoing DNA synthesis and mitosis and then are arrested in the G1 phase. Progress through the cell cycle is reinitiated after suspended cells attach to a surface. When monolayer cells in late G1 phase (just before entering S phase) are put in suspension cultures they do not initiate DNA synthesis.     

Arrest of 3T3 cells in G1 phase in suspension culture.

3T3 cells do not grow in Methocel suspension culture, while other permanent cell lines do. The viability of 3T3 cells in suspension remains unchanged for at least three days with respect to plating efficiency, vital staining and resumption of normal growth when transferred into monolayer culture. When monolayer 3T3 cells in G1 phase are suspended they remain in G1 phase. Cells already in S phase which are suspended complete ongoing DNA synthesis and mitosis and then are arrested in the G1 phase. Progress through the cell cycle is reinitiated after suspended cells attach to a surface. When monolayer cells in late G1 phase (just before entering S phase) are put in suspension cultures they do not initiate DNA synthesis.     

IQGAP1 translocates to the nucleus in early S-phase and contributes to cell cycle progression after DNA replication arrest

IQGAP1 is a plasma membrane-associated protein and an important regulator of the actin cytoskeleton, contributing to cell migration, polarity and adhesion. In this study, we demonstrate the nuclear translocation of IQGAP1 using confocal microscopy and cell fractionation. Moreover, we identify a specific pool of IQGAP1 that accumulates in the nucleus during late G1-early S phase of the cell cycle. The nuclear targeting of IQGAP1 was facilitated by N- and C-terminal sequences, and its ability to slowly shuttle between nucleus and cytoplasm/membrane was partly regulated by the CRM1 export receptor. The inhibition of GSK-3β also stimulated nuclear localization of IQGAP1. The dramatic nuclear accumulation of IQGAP1 observed when cells were arrested in G1/S phase suggested a possible role in cell cycle regulation. In support of this, we used immunoprecipitation assays to show that the nuclear pool of IQGAP1 in G1/S-arrested cells associates with DNA replication complex factors RPA32 and PCNA. More important, the siRNA-mediated silencing of IQGAP1 significantly delayed cell cycle progression through S phase and G2/M in NIH 3T3 cells released from thymidine block. Our findings reveal an unexpected regulatory pathway for IQGAP1, and show that a pool of this cytoskeletal regulator translocates into the nucleus in late G1/early S phase to stimulate DNA replication and progression of the cell cycle.     

Restoration of radiation-induced G1-depression in transformed fibroblasts treated with dextran sulfate

3T6 and 3T3 cells were cultured with dextran sulfate and irradiated with a dose of 1 000 R of ⁶⁰Co gamma-rays. The rate of progress of cells from G1 to S phase was estimated by radioautograms using ³H-thymidine as a tracer. When cultured in normal medium, 3T3 cells showed a rate of progress from G1 to S phase which was retarded by gamma-ray radiation, whereas 3T6 cells were unaffected. Dextran sulfate alone did not prolong the cell cycle time during logarithmic growth in either cell line, but reduced markedly the saturation density of 3T6 cells. Radiation-induced G1-suppression was observed in 3T6 cells which were cultured in the presence of dextran sulfate for at least 2 days. Replacement of normal media by media containing dextran sulfate at the confluent stage led to the onset of DNA synthesis (and subsequently cell division) in 3T6 cells. Gamma-ray irradiation before the change of media delayed the onset of DNA synthesis.     

Nuclear phosphoinositide 3-kinase C2beta activation during G2/M phase of the cell cycle in HL-60 cells

The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 cells blocked by aphidicolin at G(1)/S boundary and allowed to progress synchronously through the cell cycle. The activity of immunoprecipitated PI3K-C2beta in the nuclei and nuclear envelopes showed peak activity at 8 h after release from the G(1)/S block, which correlates with G(2)/M phase of the cell cycle. In the nuclei and nuclear envelopes isolated from HL-60 cells at 8 h after release from G(1)/S block, a significant increase in the level of incorporation of radiolabeled phosphate into phosphatidylinositol 3-phosphate (PtdIns(3)P) was observed with no change in the level of radiolabeled PtdIns(4)P, PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3). On Western blots, PI3K-C2beta revealed a single immunoreactive band of 180 kDa, whereas in the nuclei and nuclear envelopes isolated at 8 h after release, the gel shift of 18 kDa was observed. When nuclear envelopes were treated for 20 min with mu-calpain in vitro, the similar gel shift and increase in PI3K-C2beta activity was observed which was completely inhibited by pretreatment with calpain inhibitor calpeptin. The presence of PI3K inhibitor LY 294002 completely abolished the calpain-mediated increase in the activity of PI3K-C2beta but did not prevent the gel shift. When HL-60 cells were released from G(1)/S block in the presence of either calpeptin or LY 294002, the activation of nuclear PI3K-C2beta was completely inhibited. These results demonstrate the calpain-mediated activation of the nuclear PI3K-C2beta during G(2)/M phase of the cell cycle in HL-60 cells.     

Ribosomal protein S3 is phosphorylated by Cdk1/cdc2 during G2/M phase

Ribosomal protein S3 (rpS3) is a multifunctional protein involved in translation, DNA repair, and apoptosis. The relationship between rpS3 and cyclin-dependent kinases (Cdks) involved in cell cycle regulation is not yet known. Here, we show that rpS3 is phosphorylated by Cdk1 in G2/M phase. Co-immunoprecipitation and GST pull-down assays revealed that Cdk1 interacted with rpS3. An in vitro kinase assay showed that Cdk1 phosphorylated rpS3 protein. Phosphorylation of rpS3 increased in nocodazole-arrested mitotic cells; however, treatment with Cdk1 inhibitor or Cdk1 siRNA significantly attenuated this phosphorylation event. The phosphorylation of a mutant form of rpS3, T221A, was significantly reduced compared with wild-type rpS3. Decreased phosphorylation and nuclear accumulation of T221A was much more pronounced in G2/M phase. These results suggest that the phosphorylation of rpS3 by Cdk1 occurs at Thr221 during G2/M phase and, moreover, that this event is important for nuclear accumulation of rpS3.     

Ras-induced colony formation and anchorage-independent growth inhibited by elevated expression of Puralpha in NIH3T3 cells

Levels of Puralpha, a conserved, sequence-specific single-stranded DNA and RNA binding protein, fluctuate during the cell cycle, declining at the onset of S-phase and peaking at mitosis. In early G1 Puralpha is associated with the hypophosphorylated form of the retinoblastoma protein, Rb. Microinjection of purified Puralpha into NIH3T3 cells arrests the cell cycle at either G1/S or G2/M checkpoints with distinct morphological consequences. Here we ask whether expression of Puralpha can affect colony formation and anchorage-independent growth in ras-transformed NIH3T3 cells. Two to five-fold elevated levels of Puralpha in stably-transfected cell lines retard entry into and progression through S phase in both ras-transformed and non-transformed cells. Puralpha significantly inhibits colony formation by ras-transformed cells but not by non-transformed cells. In addition, cells transfected to express Puralpha formed only about 1/5 the number of large colonies in soft agar as control-transfected cells, demonstrating a marked inhibition of anchorage-independent growth by Puralpha. Biochemical analysis of nuclear and cytoplasmic Puralpha proteins and confocal microscopic analysis of Puralpha location indicate that access of Puralpha to the nucleus is controlled by both protein modification and sequence domains within the protein. Analyses of deletion mutants identify Puralpha domains mediating nuclear exclusion, including several potential destruction motifs and a PEST sequence at aa's 215-231. In the nucleus Puralpha colocalizes with CDK2 and cyclin A. Puralpha and cyclin D1, however, do not colocalize in the nucleus. At mitosis Puralpha is visualized about the condensed chromosomes and in the cytoplasm, where it colocalizes with cyclin B1. The data indicate that the ability of Puralpha to interact with proteins regulating cell proliferation and transformation is controlled by signals that govern its intracellular localization.