Genetic analysis of a Chinese hamster cell line lacking a G1 phase

A Chinese hamster cell line (V79-8), which lacks a measurable G1 phase in its cell cycle (G1^?), has been investigated utilizing somatic cell hybrids. The results indicate that the G1^? phenotype is dominant in two types of intraspecific hybrids and, surprisingly is in contrast recessive in two different interspecific crosses. Furthermore, the G1^? V79-8 cell line complements two different G1 defective cell cycle mutants, suggesting that V79-8 retains and can express at least some G1-specific functions.     

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.     

Heterogeneity of planarian stem cells in the S/G2/M phase

The planarian adult stem cell (pASC) population has a specific molecular signature and can be easily visualized and isolated by flow cytometry. However, the lack of antibodies against specific surface markers for planarian cells prevents a deeper analysis of specific cell populations. Here, if we describe the results of the immunoscreening of pASC plasma membrane proteins (PMPs). A novel papain-based method for planarian cell dissociation enabling both high yield and improved cell viability was used to generate single cell preparations for PMP purification. PMPs were used for intraperitoneal immunization of mice and thus about 1000 hybridoma clones were generated and screened. Supernatants collected from the hybridoma clones were first screened by ELISA and then by live immuno-staining. About half of these supernatants stained all the planarian cells, whereas the other half specifically labeled a subfraction thereof. A detailed analysis of two hybridoma supernatants revealed that large subfractions of the X1, X2 and Xin populations differentially express specific membrane markers. Quantitative PCR data disclosed a correlation between the immunostaining results and the expression of markers of the early and late progeny, also for those pASCs in the S/G2/M phase of the cell cycle (X1 population). Thus, about two thirds of the cycling pASCs showed a specific membrane signature coupled with the expression of markers hitherto considered to be restricted to differentiating, post-mitotic progeny. In summary, a library of 66 monoclonal antibodies against planarian PMPs was generated. The analysis of two of the clones generated revealed that a subset of cells of the X1 population expresses early and late progeny markers, which might indicate that these cells are committed while still proliferating. The findings demonstrate the usefulness of our PMP antibody library for planarian research.     

Two zinc-dependent steps during G1 to S phase transition

The influence of zinc (Zn) availability on thymidine kinase mRNA concentration has been investigated in cells in which production of the mRNA was regulated by either truncated thymidine kinase promoters or by the SV40 early promoter. Thymidine kinase mRNA concentrations were decreased by low Zn availability even when the promoter was truncated to 80 bp but not when it was replaced by the SV40 promoter. However, thymidine incorporation by the SV40 cells was still sensitive to lack of Zn, suggesting a second Zn-sensitive process involved in commitment to S phase. The increase in histone H3 mRNA production prior to S phase was not inhibited by lack of Zn leading to a preferential increase in this mRNA in exponentially growing cells deprived of Zn. © 1993 Wiley-Liss, Inc.     

G(1) to S transition: more than a cell cycle engine switch

CDK-cyclin complexes are the universal drivers of cell cycle transitions. Progression through G(1) and transition to S-phase, thereby initiating genome duplication, requires the concerted action of cyclin-dependent kinase (CDK)-cyclin complexes on specific targets. These targets belong to at least two major regulatory networks: the retinoblastoma-related (RBR)/E2F pathway and complexes that are responsible for the initiation of DNA replication. The G(1) phase is central to the integration of signals that regulate both the exit from the cell division cycle to differentiation and the reactivation of cell proliferation. Cellular factors that are involved in these pathways play a role in regulating cell size and number, and organogenesis. As a consequence, they are also involved in determining plant architecture.     

Indomethacin-induced G1/S phase arrest of the plant cell cycle

In animal systems, indomethacin inhibits cAMP production via a prostaglandin-adenylyl cyclase pathway. To examine the possibility that a similar mechanism occurs in plants, the effect of indomethacin on the cell cycle of a tobacco bright yellow 2 (TBY-2) cell suspension was studied. Application of indomethacin during mitosis did not interfere with the M/G1 progression in synchronized BY-2 cells but it inhibited cAMP production at the beginning of the G1 phase and arrested the cell cycle progression at G1/S. These observations are discussed in relation to the putative involvement of cAMP biosynthesis in the cell cycle progression in TBY-2 cells.     

Saccharomyces cerevisiae lacking Snm1, Rev3 or Rad51 have a normal S-phase but arrest permanently in G2 after cisplatin treatment

The role of Snm1, Rev3 and Rad51 in S-phase after cisplatin (CDDP) DNA treatment has been examined. When isogenic deletion mutants snm1 delta, rev3 delta and rad51 delta were arrested in G1 and treated with doses of CDDP causing significant lethality (<20% survival in the mutant strains), they progressed through S-phase with normal kinetics. The mutants arrested in G2 like wild-type cells, however they did not exit the arrest and reenter the cell cycle. This finding demonstrates that these genes are not required to allow DNA replication in the presence of damage. Therefore, Snm1, Rev3 and Rad51 may act after S to allow repair. At high levels of damage (<40% survival in wild-type cells) S-phase was slowed in a MEC1-dependent fashion. The cross-link incision kinetics of snm1 delta and rev3 delta mutants were also examined; both showed no deficiencies in incision of cross-linked DNA.     

A new method to discriminate G1, S, G2, M, and G1 postmitotic cells

A new flow cytometric method combining light scattering measurements, detection of bromodeoxyuridine (BrdU) incorporation via fluorescent antibody, and quantitation of cellular DNA content by propidium iodide (PI) allows identification of additional compartments in the cell cycle. Thus, while cell staining with BrdU-antibodies and PI reveals the G1, S, and G2 + M phases of the cell cycle, differences in light scattering allow separation of G2 phase cells from M phase cells and subdivision of G1 phase into two compartments, i.e., G1A representing postmitotic cells which mature to G1B cells ready to initiate DNA synthesis. The method involves fixation of cells in 70% ethanol, extraction of histones with HC1, and thermal denaturation of DNA. This treatment appears to enhance the differences in chromatin structure of cells in the various phases of the cell cycle to the extent that cells could be separated on the basis of the 90 degrees scatter. Mitotic cells show much lower scatter than G2 phase cells, and G1 postmitotic cells (G1A) show lower scatter than G1 cells about to enter the S phase (G1B). Light scattering is correlated with chromatin condensation, as judged by microscopic evaluation of cells sorted on the basis of light scatter. The method has the advantage over the parental BrdU/DNA bivariate analysis in allowing the G2 and M phases of the cell cycle to be separated and the G1 phase to be analyzed in more detail. The method may also allow separation of unlabeled S phase cells from mitotic cells and distinguish between labeled and unlabeled mitotic cells.     

Cdc2-cyclin E complexes regulate the G1/S phase transition

The cyclin-dependent kinase inhibitor p27(Kip1) is known as a negative regulator of cell-cycle progression and as a tumour suppressor. Cdk2 is the main target of p27 (refs 2, 3) and therefore we hypothesized that loss of Cdk2 activity should modify the p27(-/-) mouse phenotype. Here, we show that although p27(-/-) Cdk2(-/-) mice developed ovary tumours and tumours in the anterior lobe of the pituitary, we failed to detect any functional complementation in p27(-/-) Cdk2(-/-) double-knockout mice, indicating a parallel pathway regulated by p27. We observed elevated levels of S phase and mitosis in tissues of p27(-/-) Cdk2(-/-) mice concomitantly with elevated Cdc2 activity in p27(-/-) Cdk2(-/-) extracts. p27 binds to Cdc2, cyclin B1, cyclin A2, or suc1 complexes in wild-type and Cdk2(-/-) extracts. In addition, cyclin E binds to and activates Cdc2. Our in vivo results provide strong evidence that Cdc2 may compensate the loss of Cdk2 function.     

Mice lacking c-fos have normal hematopoietic stem cells but exhibit altered B-cell differentiation due to an impaired bone marrow environment.

Mice lacking c-fos develop severe osteopetrosis with deficiencies in bone remodeling and exhibit extramedullary hematopoiesis, thymic atrophy, and altered B-cell development. In this study, we have used these mice to characterize in detail the developmental potential of hematopoietic stem cells lacking c-fos and to analyze how the lymphoid differentiation is altered. In c-fos -/- mice, B-cell numbers are reduced in the spleen, lymph nodes, and the peripheral blood as a result of a marked reduction (> 90%) in the number of clonogenic B-cell precursors. In contrast, the number and lineage distribution of myeloid progenitor cells are not affected. The thymic defects observed in a large number of these mice correlate with their health status, suggesting that this may be an indirect effect of the c-fos mutation. In vitro differentiation and bone marrow reconstitution experiments demonstrated that hematopoietic stem cells lacking c-fos can give rise to all mature myeloid as well as lymphoid cells, suggesting that the observed B lymphopenia in the mutant mice is due to an altered environment. Transplantation of wild-type bone marrow cells into newborn mutant mice resulted in the establishment of a bone marrow space and subsequent correction of the B-cell defect. These results demonstrate that hematopoietic stem cells lacking Fos have full developmental potential and that the observed defect in B-cell development is most likely due to the impaired bone marrow environment as a consequence of osteopetrosis.     

A mesoscale model of G1/S phase transition in liver regeneration

The liver regenerates and maintains its function and size after injury by counterbalancing cell death with compensatory cell division. During liver regeneration, injured sites release cytokines, which stimulate normally quiescent hepatocytes to re-enter cell division cycle. Using a mesoscale approach, we have implemented the first mathematical model that describes cytokine-induced dedifferentiation of hepatocytes and the subsequent initiation of DNA synthesis (G0/G1 and G1/S phase transitions of the cell cycle). The model accurately reproduces experimentally measured kinetics of various signaling intermediates and DNA synthesis in hepatocytes for varying degrees of liver damage, in both wild type and knockout backgrounds. Liver regeneration is known to be a robust process, as liver mass reconstitution still occurs in various knockout mice (albeit with different kinetics). We analyze the robustness of the model using methods of control analysis. Moreover, we discuss the system's bandpass filtering properties and delays, which arise from feedbacks and nested feed-forward loops.     

Human Mcm proteins at a replication origin during the G1 to S phase transition

Previous work with yeast cells and with Xenopus egg extracts had shown that eukaryotic pre-replication complexes assemble on chromatin in a step-wise manner whereby specific loading factors promote the recruitment of essential Mcm proteins at pre-bound origin recognition complexes (ORC with proteins Orc1p–Orc6p). While the order of assembly—Mcm binding follows ORC binding—seems to be conserved in cycling mammalian cells in culture, it has not been determined whether mammalian Mcm proteins associate with ORC-bearing chromatin sites. We have used a chromatin immunoprecipitation approach to investigate the site of Mcm binding in a genomic region that has previously been shown to contain an ORC-binding site and an origin of replication. Using chromatin from HeLa cells in G₁ phase, antibodies against Orc2p as well as antibodies against Mcm proteins specifically immunoprecipitate chromatin enriched for a DNA region that includes a replication origin. However, with chromatin from cells in S phase, only Orc2p-specific antibodies immunoprecipitate the origin-containing DNA region while Mcm-specific antibodies immunoprecipitate chromatin with DNA from all parts of the genomic region investigated. Thus, human Mcm proteins first assemble at or adjacent to bound ORC and move to other sites during genome replication.     

The tumour suppressor gene product APC blocks cell cycle progression from G0/G1 to S phase.

The APC gene is mutated in familial adenomatous polyposis (FAP) as well as in sporadic colorectal tumours. The product of the APC gene is a 300 kDa cytoplasmic protein associated with the adherence junction protein catenin. Here we show that overexpression of APC blocks serum-induced cell cycle progression from G0/G1 to the S phase. Mutant APCs identified in FAP and/or colorectal tumours were less inhibitory and partially obstructed the activity of the normal APC. The cell-cycle blocking activity of APC was alleviated by the overexpression of cyclin E/CDK2 or cyclin D1/CDK4. Consistent with this result, kinase activity of CDK2 was significantly down-regulated in cells overexpressing APC although its synthesis remained unchanged, while CDK4 activity was barely affected. These results suggest that APC may play a role in the regulation of the cell cycle by negatively modulating the activity of cyclin-CDK complexes.     

Polyamine biosynthesis and accumulation during the G1 to S phase transition

Ornithine decarboxylase, an important enzyme in growth regulation, is increased in CHO cells in G₁ phase of the cell cycle and decreases as the cells progress into S phase. S-adenosyl-L-methionine decarboxylase activity, which is dependent on either the presence of putrescine or spermidine for the synthesis of spermidine and spermine respectively, shows a maximal increase in late G₁/early S phase which corresponds very closely with the cell cycle phase specific accumulation of spermidine and spermine during S phase. Total culture evaluation of spermidine and spermine, which included extracellular as well as intracellular concentrations, indicated that extracellular accumulations of these polyamines occurred only in G₁ and that entry into S phase was concomitant with intracellular accumulation patterns. Hyperthermia (43°C for 1 hour) in mid-G₁ phase of the cell cycle resulted in rapid decreases in the activities of ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase. In these cells, DNA replication was also not detectable until nine hours after mitosis, a time at which there had been recovery of ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase activities. Previous data have further indicated a requirement for polyamine reaccumulation before control DNA replication rates are resumed. We therefore suggest that polyamine biosynthesis and intracellular accumulation are both temporal and quantitative prerequisites for transition through S phase.     

G1/S and G2/M Cyclin-Dependent Kinase Activities Commit Cells to Death in the Absence of the S-Phase Checkpoint

The Mec1 and Rad53 protein kinases are essential for budding yeast cell viability and are also required to activate the S-phase checkpoint, which supports DNA replication under stress conditions. Whether these two functions are related to each other remains to be determined, and the nature of the replication stress-dependent lethality of mec1 and rad53 mutants is still unclear. We show here that a decrease in cyclin-dependent kinase 1 (Cdk1) activity alleviates the lethal effects of mec1 and rad53 mutations both in the absence and in the presence of replication stress, indicating that the execution of a certain Cdk1-mediated event(s) is detrimental in the absence of Mec1 and Rad53. This lethality involves Cdk1 functions in both G₁ and mitosis. In fact, delaying either the G₁/S transition or spindle elongation in mec1 and rad53 mutants allows their survival both after exposure to hydroxyurea and under unperturbed conditions. Altogether, our studies indicate that inappropriate entry into S phase and segregation of incompletely replicated chromosomes contribute to cell death when the S-phase checkpoint is not functional. Moreover, these findings suggest that the essential function of Mec1 and Rad53 is not necessarily separated from the function of these kinases in supporting DNA synthesis under stress conditions.     

Cell size at S phase initiation: an emergent property of the G1/S network

The eukaryotic cell cycle is the repeated sequence of events that enable the division of a cell into two daughter cells. It is divided into four phases: G₁, S, G₂, and M. Passage through the cell cycle is strictly regulated by a molecular interaction network, which involves the periodic synthesis and destruction of cyclins that bind and activate cyclin-dependent kinases that are present in nonlimiting amounts. Cyclin-dependent kinase inhibitors contribute to cell cycle control. Budding yeast is an established model organism for cell cycle studies, and several mathematical models have been proposed for its cell cycle. An area of major relevance in cell cycle control is the G₁ to S transition. In any given growth condition, it is characterized by the requirement of a specific, critical cell size, P_S, to enter S phase. The molecular basis of this control is still under discussion. The authors report a mathematical model of the G₁ to S network that newly takes into account nucleo/cytoplasmic localization, the role of the cyclin-dependent kinase Sic1 in facilitating nuclear import of its cognate Cdk1-Clb5, Whi5 control, and carbon source regulation of Sic1 and Sic1-containing complexes. The model was implemented by a set of ordinary differential equations that describe the temporal change of the concentration of the involved proteins and protein complexes. The model was tested by simulation in several genetic and nutritional setups and was found to be neatly consistent with experimental data. To estimate P_S, the authors developed a hybrid model including a probabilistic component for firing of DNA replication origins. Sensitivity analysis of P_S provides a novel relevant conclusion: P_S is an emergent property of the G₁ to S network that strongly depends on growth rate.     

The slow S to M fluorescence rise in cyanobacteria is due to a state 2 to state 1 transition

In dark-adapted plants and algae, chlorophyll a fluorescence induction peaks within 1s after irradiation due to well documented photochemical and non-photochemical processes. Here we show that the much slower fluorescence rise in cyanobacteria (the so-called "S to M rise" in tens of seconds) is due to state 2 to state 1 transition. This has been demonstrated in particular for Synechocystis PCC6803, using its RpaC(-) mutant (locked in state 1) and its wild-type cells kept in hyperosmotic suspension (locked in state 2). In both cases, the inhibition of state changes correlates with the disappearance of the S to M fluorescence rise, confirming its assignment to the state 2 to state 1 transition. The general physiological relevance of the SM rise is supported by its occurrence in several cyanobacterial strains: Synechococcus (PCC 7942, WH 5701) and diazotrophic single cell cyanobacterium (Cyanothece sp. ATCC 51142). We also show here that the SM fluorescence rise, and also the state transition changes are less prominent in filamentous diazotrophic cyanobacterium Nostoc sp. (PCC 7120) and absent in phycobilisome-less cyanobacterium Prochlorococcus marinus PCC 9511. Surprisingly, it is also absent in the phycobiliprotein rod containing Acaryochloris marina (MBIC 11017). All these results show that the S to M fluorescence rise reflects state 2 to state 1 transition in cyanobacteria with phycobilisomes formed by rods and core parts. We show that the pronounced SM fluorescence rise may reflect a protective mechanism for excess energy dissipation in those cyanobacteria (e.g. in Synechococcus PCC 7942) that are less efficient in other protective mechanisms, such as blue light induced non-photochemical quenching. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Nuclear phospholipase C-beta1b activation during G2/M and late G1 phase in nocodazole-synchronized HL-60 cells

In this study, the activity of nuclear phosphatidylinositol-specific phosholipase C (PI-PLC) was investigated in HL-60 cells blocked at G(2)/M phase by the addition of nocodazole, and released into medium as synchronously progressing cells. Two peaks of an increase in the nuclear PI-PLC activities were detected; an early peak reached a maximum at 1 h after release from the nocodazole block, and a second increase was detected at 8.5 h after the release. Immunoprecipitation studies indicated that the increase in the activity was due to the activation of the nuclear PI-PLC-beta(1). Western blot analysis demonstrated no changes in the level of both a and b splicing variants of PI-PLC-beta(1) in the nuclei of cells isolated at either 1 h or 8.5 h after the block. However, an increase in the serine-phosphorylation of PI-PLC-beta(1b) was detected in the nuclei of HL-60 cells isolated at 1 and 8.5 h after the block, and the presence of MEK-inhibitor PD98059 completely inhibited both the serine phosphorylation and the increase in the PI-PLC activities in vitro. The presence of PI-PLC inhibitor prevented the progression of HL-60 cells through the G(1) into S phase of the cell cycle. These results demonstrate that two peaks of nuclear PI-PLC activities, which are due to a PD98059-sensitive phosphorylation of nuclear PLC-beta(1b) on serine, occur at the G(2)/M and late G(1) phase and are necessary for the progression of the cells through the cell cycle.