Reversible G1 arrest of a human lung epithelial cell line by staurosporine.

Staurosporine, a microbial-derived protein kinase inhibitor, reversibly blocked non-synchronized, replicating cultures of the human lung epithelial cell line EKVX in the G1 phase of cell cycle and inhibited DNA synthesis and cell replication. The mechanism of this cell-cycle arrest in EKVX cells by staurosporine was likely due to inhibition of protein kinase C (PKC) because: 1) dose-dependent inhibition of DNA synthesis occurred at levels of staurosporine that inhibit phosphorylation of PKC substrate, 2) inhibition of DNA synthesis was also seen after treatment with another PKC inhibitor H7, but not by the chemically similar HA1004, which has a relative inhibitory specificity for cAMP-dependent protein kinase, and 3) the DNA synthesis was not inhibited by specific tyrosine kinase inhibitors Genistein and Lavendustin A at concentrations that inhibit tyrosine kinase activity. Removal of staurosporine from cell culture media resulted in a rebound in PKC activity and synchronized DNA synthesis in EKVX cultures. The reversibility of the inhibition was noted even after 5 days of treatment with staurosporine, and DNA synthesis remained synchronized for at least two rounds of cell replication after removal of staurosporine. Flow cytometric analysis confirmed that more than 90% of the cell population was blocked in the G1 phase after cells were treated with staurosporine for 24 h. Agents such as staurosporine may be useful for synchronizing cell populations to study cell-cycle specific biochemical events important for the regulation of cell replication in the EKVX cell line.     

Chromatin structure restricts origin utilization when quiescent cells re-enter the cell cycle

Quiescent cells reside in G0 phase, which is characterized by the absence of cell growth and proliferation. These cells remain viable and re-enter the cell cycle when prompted by appropriate signals. Using a budding yeast model of cellular quiescence, we investigated the program that initiated DNA replication when these G0 cells resumed growth. Quiescent cells contained very low levels of replication initiation factors, and their entry into S phase was delayed until these factors were re-synthesized. A longer S phase in these cells correlated with the activation of fewer origins of replication compared to G1 cells. The chromatin structure around inactive origins in G0 cells showed increased H3 occupancy and decreased nucleosome positioning compared to the same origins in G1 cells, inhibiting the origin binding of the Mcm4 subunit of the MCM licensing factor. Thus, quiescent yeast cells are under-licensed during their re-entry into S phase.     

The concentration-dependent diversity of effects of DNA topoisomerase I and II inhibitors on the cell cycle of HL-60 cells.

Exposure of promyelocytic leukemic HL-60 cells to 3-60 nM of the DNA topoisomerase I inhibitor camptothecin (CAM) or to 30-450 nM and 0.12-1.5 microM of DNA topoisomerase II inhibitors teniposide (TN) and 4-(9-acridynylamino)-3-methanesulfon-m-anisidide (m-AMSA), respectively, resulted in two distinct kinetic effects: (1) the cells entered S phase but the rate of DNA replication was reduced in proportion to the inhibitor concentration; (2) the transition from G2 to M was impaired, approximately 1 h after addition of the inhibitor. As a consequence, the cells accumulated in the S (preferentially in early S) and in G2 phases of the cell cycle. Whereas CAM was more efficient in suppressing cell progression through S phase, TN and m-AMSA were more potent G2 blockers. At these low inhibitor concentrations no signs of immediate cytotoxicity or DNA degradation were apparent. However, above 145 nM of CAM, 900 nM of TN, or 2 microM of m-AMSA extensive DNA degradation in nuclei of S phase cells was evident within 6 h of addition of the inhibitor, resulting in the loss of S and G2 + M cells from these cultures. The data indicate that depending on concentration, mechanisms mediating the cytostatic/cytotoxic activity of both DNA topoisomerase I and II inhibitors may be quite different. Suppression of the DNA replication and the G2 to M transition, seen at low inhibitor concentrations, is compatible with the assumption that the inhibitor-induced stabilization of the topoisomerase-DNA cleavable complexes interferes with DNA replication and chromosome condensation/segregation, respectively. Above the threshold concentration for each inhibitor, an endonucleolytic activity is triggered, resulting in rapid DNA degradation in nuclei of S and G2 phase cells. The endonucleolytic effect is not only cell cycle phase-specific but is also modulated by tissue-specific factors because it cannot be observed, e.g., in the lymphocytic leukemic cell lines.     

Activation of SV40 DNA replication in vitro by cellular protein phosphatase 2A.

We have made use of the cell-free SV40 DNA replication system to identify and characterize cellular proteins required for efficient DNA synthesis. One such protein, replication protein C (RP-C), was shown to be involved with SV40 large T antigen in the early stages of viral DNA replication in vitro. We demonstrate here that RP-C is identical to the catalytic subunit of cellular protein phosphatase 2A (PP2Ac). The purified protein dephosphorylates specific phosphoamino acid residues in T antigen, consistent with the hypothesis that SV40 DNA replication is regulated by modulating the phosphorylation state of the viral initiator protein. We also show that purified RP-C/PP2Ac preferentially stimulates SV40 DNA replication in extracts from early G1 phase cells. This finding suggests that the activity of a cellular factor that influences the net phosphorylation state of T antigen is cell cycle dependent.     

A general protocol for evaluating the specific effects of DNA replication inhibitors.

Inhibitors of DNA replication in mammalian cells are of great interest because of their potential use in chemotherapy and in cell synchronizing protocols in the laboratory. We have used a combination of isotopic labelling protocols and a two-dimensional gel replicon mapping procedure to determine the specific effects of five different replication inhibitors in cultured cells. Utilizing this protocol, we show that hydroxyurea, aphidicolin, and cytosine arabinoside, three known chain elongation inhibitors, are rather ineffective at preventing fork progression even at relatively high concentrations. In contrast, two related compounds that have been suggested to be G1/S inhibitors (mimosine and ciclopyrox olamine [CPX]) actually appear to inhibit initiation at origins. One of these agents (CPX) appears also to inhibit replication in yeast, opening the possibility that the gene encoding the target (initiator?) protein can first be identified in yeast by genetic approaches and can then be used to isolate the mammalian homologue.     

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.     

Determination of growth inhibitory action point of interferon gamma on WISH cells in cell cycle progression and the window of responsiveness of the cells to the interferon

We had earlier shown that human foetal epithelial cells (WISH), growth-inhibited by interferon gamma (IFNgamma), were reversibly detained at a point prior to DNA synthesis. In the present study, we determined the window of action of IFNgamma in the G1 phase duration and the exact point of detention of WISH cells in cell cycle progression with respect to the known points of detention by the inhibitors of DNA replication initiation (aphidicolin and carbonyl diphosphonate) and of activation of replication protein A (6-dimethylaminopurine), of which RPA activation being the earlier event compared to DNA replication initiation in cell cycle progression. WISH cells, which were released from IFNgamma-induced arrest, permeabilised and exposed independently to these inhibitors show that IFNgamma detains WISH cells prior to initiation of DNA synthesis. Further, exposure of IFNalpha-synchronized (at G0/G1) or mimosine-synchronized (at G1/S) WISH cells to IFNgamma, which was added at different time points post-release from the synchronizing agent, showed that the cells were promptly responsive to the growth inhibitory action of IFNgamma only during the first 11h in G1 phase. Taken together, these results suggest that IFNgamma inhibits growth of WISH cells by detaining them at a point prior to initiation of DNA synthesis and that the IFN acts within the first 11h in G1 phase of the cell cycle.     

The cell cycle regulator p27Kip1 interacts with MCM7, a DNA replication licensing factor, to inhibit initiation of DNA replication

The G1/S phase restriction point is a critical checkpoint that interfaces between the cell cycle regulatory machinery and DNA replicator proteins. Here, we report a novel function for the cyclin-dependent kinase inhibitor p27Kip1 in inhibiting DNA replication through its interaction with MCM7, a DNA replication protein that is essential for initiation of DNA replication and maintenance of genomic integrity. We find that p27Kip1 binds the conserved minichromosome maintenance (MCM) domain of MCM7. The proteins interact endogenously in vivo in a growth factor-dependent manner, such that the carboxyl terminal domain of p27Kip1 inhibits DNA replication independent of its function as a cyclin-dependent kinase inhibitor. This novel function of p27Kip1 may prevent inappropriate initiation of DNA replication prior to S phase.     

Mechanism in the sequential control of cell morphology and S phase entry by epidermal growth factor involves distinct MEK/ERK activations

Cell shape plays a role in cell growth, differentiation, and death. Herein, we used the hepatocyte, a normal, highly differentiated cell characterized by a long G1 phase, to understand the mechanisms that link cell shape to growth. First, evidence was provided that the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) cascade is a key transduction pathway controlling the hepatocyte morphology. MEK2/ERK2 activation in early G1 phase did not lead to cell proliferation but induced cell shape spreading and demonstration was provided that this MAPK-dependent spreading was required for reaching G1/S transition and DNA replication. Moreover, epidermal growth factor (EGF) was found to control this morphogenic signal in addition to its mitogenic effect. Thus, blockade of cell spreading by cytochalasin D or PD98059 treatment resulted in inhibition of EGF-dependent DNA replication. Our data led us to assess the first third of G1, is exclusively devoted to the growth factor-dependent morphogenic events, whereas the mitogenic signal occurred at only approximately mid-G1 phase. Moreover, these two growth factor-related sequential signaling events involved successively activation of MEK2-ERK2 and then MEK1/2-ERK1/2 isoforms. In addition, we demonstrated that inhibition of extracellular matrix receptor, such as integrin beta1 subunit, leads to cell arrest in G1, whereas EGF was found to up-regulated integrin beta1 and fibronectin in a MEK-ERK-dependent manner. This process in relation to cytoskeletal reorganization could induce hepatocyte spreading, making them permissive for DNA replication. Our results provide new insight into the mechanisms by which a growth factor can temporally control dual morphogenic and mitogenic signals during the G1 phase.     

Geldanamycin induces cell cycle arrest in K562 erythroleukemic cells

Geldanamycin (GA), a benzoquinone ansamycin, is one of the specific inhibitors of 90-kDa heat shock protein and induces growth inhibition and apoptosis in certain cancer cell lines. We have investigated the mechanism of GA-induced growth inhibition in K562 erythroleukemic cells. DNA flow-cytometric analysis indicated that GA-induced growth arrest was associated with G2/M phase arrest of the cell cycle. GA treatment down-regulated the expression of cyclin B1 and inhibited phosphorylation of Cdc2 protein, both key regulatory proteins at the G2/M boundary. GA also markedly inhibited the Cdc2 kinase activity, which may be in part a result of up-regulation of p27KIP1 by GA. The present results suggest a novel mechanism that p27KIP1 could be involved in the regulation of G2 to M phase transition.     

CDK4/6 inhibitors induce replication stress to cause long‐term cell cycle withdrawal

CDK4/6 inhibitors arrest the cell cycle in G1‐phase. They are approved to treat breast cancer and are also undergoing clinical trials against a range of other tumour types. To facilitate these efforts, it is important to understand why a cytostatic arrest in G1 causes long‐lasting effects on tumour growth. Here, we demonstrate that a prolonged G1 arrest following CDK4/6 inhibition downregulates replisome components and impairs origin licencing. Upon release from that arrest, many cells fail to complete DNA replication and exit the cell cycle in a p53‐dependent manner. If cells fail to withdraw from the cell cycle following DNA replication problems, they enter mitosis and missegregate chromosomes causing excessive DNA damage, which further limits their proliferative potential. These effects are observed in a range of tumour types, including breast cancer, implying that genotoxic stress is a common outcome of CDK4/6 inhibition. This unanticipated ability of CDK4/6 inhibitors to induce DNA damage now provides a rationale to better predict responsive tumour types and effective combination therapies, as demonstrated by the fact that CDK4/6 inhibition induces sensitivity to chemotherapeutics that also cause replication stress.     

Spontaneous mutation of leu2 in Saccharomyces cerevisiae

The data obtained indicate that spontaneous mutations in Saccharomyces cerevisiae are formed during DNA replication. With no DNA replication in the lag-period, in the stationary growth phase, spontaneous mutations are not formed in cell culture during the G1 phase of cell cycle. Experimental data show the absence of primary spontaneously occurring DNA lesion accumulation in the cell G1 phase. Spontaneous mutations of yeasts are formed in the S phase of cell cycle, apparently as DNA replication errors. It is established that the frequency of spontaneous reversions of the leu2 gene in Saccharomyces cerevisiae strain NA3-24 increases when the cells are cultivated on the culture medium with different concentrations of leucine.     

The logic of the Membrane, Magnesium, Mitosis (MMM) model for the regulation of animal cell proliferation

The addition of animal serum or specific protein growth factors to quiescent, serum-deprived vertebrate cells in culture activates a wide variety of biochemical responses within minutes, which are followed in 5-10h by the initiation of DNA synthesis and then by mitosis. The quintessential early and continuing activation step for the increase in DNA synthesis is the increased initiation rate of protein synthesis, which must be continuously maintained throughout the G1 phase for advancement into S. The aggregate of biochemical reactions to growth factors is called the coordinate response, to indicate that many related and unrelated processes are orchestrated to repetitively reproduce cells. It is, however, crucial to recognize that the coordinate response can be induced for one or more rounds of replication by a variety of non-specific and quasi-specific membrane effectors. The logic of considering this framework of events in growth control implied that a single multi-target second messenger plays a central role in coordinating the events of the overall response. The same reasoning suggested that free Mg(2+) is the unifying regulatory element in that response which includes protein kinase pathways, and that the cytoplasmic activity of Mg(2+) increases with the binding of growth factors to their receptors in the cell membrane, or of less specific perturbations of the membrane. Experimental support of this conclusion is presented here and is represented in the MMM model of cell proliferation control.     

细胞周期研究的新进展

细胞周期研究的新进展陆长德（中国科学院上海生物化学研究所２０００３１）主要来自三方面的研究以及它们之间的相互交叉对于细胞周期研究的进展起了很大的作用。十多年来酵母分子遗传学的研究鉴定了许多与细胞周期的控制有关的基因,提供了许多突变株（如ＣＤＣ）;１９８８年对蛙卵成熟促进因子ＭＰＦ成分的鉴定和对它生物学功能的确定使人们对细胞周期的认识有了一个飞跃;人类的致癌基因（如Ｔａｇ）,肿瘤抑制基因（如p53,pRB)以及其他一些疾病(如对电离辐射敏感的遗传病,AT的分子机制的研究也大大地促进了细胞周期的研究。     

DNA damage bypass operates in the S and G2 phases of the cell cycle and exhibits differential mutagenicity

Translesion DNA synthesis (TLS) employs low-fidelity DNA polymerases to bypass replication-blocking lesions, and being associated with chromosomal replication was presumed to occur in the S phase of the cell cycle. Using immunostaining with anti-replication protein A antibodies, we show that in UV-irradiated mammalian cells, chromosomal single-stranded gaps formed in S phase during replication persist into the G2 phase of the cell cycle, where their repair is completed depending on DNA polymerase ζ and Rev1. Analysis of TLS using a high-resolution gapped-plasmid assay system in cell populations enriched by centrifugal elutriation for specific cell cycle phases showed that TLS operates both in S and G2. Moreover, the mutagenic specificity of TLS in G2 was different from S, and in some cases overall mutation frequency was higher. These results suggest that TLS repair of single-stranded gaps caused by DNA lesions can lag behind chromosomal replication, is separable from it, and occurs both in the S and G2 phases of the cell cycle. Such a mechanism may function to maintain efficient replication, which can progress despite the presence of DNA lesions, with TLS lagging behind and patching regions of discontinuity.     

The cell cycle and DNA mismatch repair

The DNA mismatch repair (MMR) pathway contributes to the fidelity of DNA synthesis and recombination by correcting mispaired nucleotides and insertion/deletion loops (IDLs). We have investigated whether MMR protein expression, activity, and subcellular location are altered during discrete phases of the cell cycle in mammalian cells. Two distinct methods have been used to demonstrate that although physiological MMR protein expression, mismatch binding, and nick-directed MMR activity within the nucleus are at highest levels during S phase, MMR is active throughout the cell cycle. Despite equal MMR nuclear protein concentrations in S and G(2) phases, mismatch binding and repair activities within G(2) are significantly lower, indicating a post-translational decrease in MMR activity specific to G(2). We further demonstrate that typical co-localization of MutSalpha to late S phase replication foci can be disrupted by 2 microM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This concentration of MNNG does not decrease ongoing DNA synthesis nor induce cell cycle arrest until the second cell cycle, with long-term colony survival decreased by only 24%. These results suggest that low level alkylation damage can selectively disrupt MMR proofreading activity during DNA synthesis and potentially increase mutation frequency within surviving cells.     

Coupling the initiation of chromosome replication to cell size in Escherichia coli

Bacterial cells change size dramatically with change in growth rate, but the ratio between cell volume and the number of copies of the origin of chromosome replication (oriC) is roughly constant at the time of initiation of DNA replication at almost all growth rates. Recent research on the inactivation of initiator protein (DnaA) and depletion of DnaA pools by the high-affinity DnaA-binding locus datA allows us to propose a simple model to explain the long-standing question of how Escherichia coli couples DNA replication to cell size.     

Synchronization in the cell cycle by inhibitors of DNA replication induces histone H2AX phosphorylation: an indication of DNA damage

Several methods to synchronize cultured cells in the cell cycle are based on temporary inhibition of DNA replication. Previously it has been reported that cells synchronized this way exhibited significant growth imbalance and unscheduled expression of cyclins A and B1. We have now observed that HL-60 cells exposed to inhibitors of DNA replication (thymidine, aphidicolin and hydroxyurea), at concentrations commonly used to synchronize cell populations, had histone H2AX phosphorylated on Ser-139. This modification of H2AX, a marker of DNA damage (induction of DNA double-strand breaks; DSBs), was most pronounced in S-phase cells, and led to their apoptosis. Thus, to a large extent, synchronization was caused by selective kill of DNA replicating cells through induction of replication stress. In fact, similar synchronization has been achieved by exposure of cells to the DNA topoisomerase I inhibitor camptothecin, a cytotoxic drug known to target S-phase cells. A large proportion of the surviving cells 'synchronized' by DNA replication inhibitors at the G1/S boundary had phosphorylated histone H2AX. Inhibitors of DNA replication, thus, not only selectively kill DNA replicating cells, induce growth imbalance and alter the machinery regulating progression through the cycle, but they also cause DNA damage involving formation of DSBs in the surviving ('synchronized') cells. The above effects should be taken into account when interpreting data obtained with the use of cells synchronized by inhibitors of DNA replication.