Calcium levels during cell cycle correlate with cell fate of Dictyostelium discoideum

Levels of intracellular calcium, (Ca(2+))(i), from different stages of cell cycle of Dictyostelium discoideum were monitored using the fluorescent Ca(2+)-sensitive dye, Indo 1. Combinations of Ca(2+)-ionophore (A23187) and Ca(2+)-chelator (EGTA) resulted in the inhibition of progression of cell cycle. This delay was caused due to block in G(2)/M-->S phase transition of the cell cycle. Rescue of the cell cycle progression was made with 0.5 m m of exogenous Ca(2+). High (Ca(2+))(i)levels overlapped with the S-phase, of the cell cycle.Results indicate that a high (Ca(2+))(i)level during S-phase is not required for cell cycle progression but for cell-type choice mechanism at the onset of starvation, and these cells tend to follow the prestalk pathway.     

Amino acid and manganese supplementation modulates the glycosylation state of erythropoietin in a CHO culture system

The manufacture of secreted proteins is complicated by the need for both high levels of expression and appropriate processing of the nascent polypeptide. For glycoproteins, such as erythropoietin (EPO), posttranslational processing involves the addition of oligosaccharide chains. We initially noted that a subset of the amino acids present in the cell culture media had become depleted by cellular metabolism during the last harvest cycle in our batch fed system and hypothesized that by supplementing these nutrients we would improve EPO yields. By increasing the concentration of these amino acids we increased recombinant human erythropoietin (rHuEPO) biosynthesis in the last harvest cycle as expected but, surprisingly, we also observed a large increase in the amount of rHuEPO with a relatively low sialic acid content. To understand the nature of this process we isolated and characterized the lower sialylated rHuEPO pool. Decreased sialylation correlated with an increase in N-linked carbohydrates missing terminal galactose moieties, suggesting that beta-1,4-galactosyltransferase may be rate limiting in our system. To test this hypothesis we supplemented our cultures with varying concentrations of manganese (Mn(2+)), a cofactor for beta-1,4-galactosyltransferase. Consistent with our hypothesis we found that Mn(2+) addition improved galactosylation and greatly reduced the amount of rHuEPO in the lower sialylated fraction. Additionally, we found that Mn(2+) addition increased carbohydrate site occupancy and narrowed carbohydrate branching to bi-antennary structures in these lower sialylated pools. Surprisingly Mn(2+) only had this effect late in the culture process. These data indicate that the addition of Mn(2+) has complex effects on stressed batch fed cultures.     

Relationship between double fertilization and the cell cycle in male and female gametes of tobacco

Nuclear DNA content of male and female gametes of tobacco was determined using 4,6-diamindino-2-phenylindole and quantitative microfluorimetry. Pollen grains are released with generative cells containing 2C DNA. Mitotic division occurs in the pollen tube 8–12 h after germination. The resulting sperm cells have 1C DNA content during pollen tube elongation in the style. Sperm cells deposited in the degenerated synergid have a DNA content between 1C and 2C, indicating that sperm are in S-phase in the synergid. Concomitant with pollen tube arrival, the egg cell increases in DNA quantity from 1C to between 1C and 2C at 48 h after pollination. In the absence of pollination, S-phase in the egg cell is delayed by up to 36 h. Newly formed zygotes contain nuclear DNA concentrations of 4C at karyogamy and remain at 4C until zygote division. Tobacco displays cell fusion after the completion of S-phase, apparently during G₂. Failure to achieve an optimized system for in vitro fertilization in Nicotiana may reflect the challenges of achieving cell cycle synchrony in gametes isolated from pollen tubes. Receptive gametes are presumably those that pass through the protracted S-phase, reaching G₂ receptivity and cell cycle congruity before fusion.     

GSK3 inhibitors stabilize Wee1 and reduce cerebellar granule cell progenitor proliferation

Ubiquitin mediated proteolysis is required for transition from one cell cycle phase to another. For instance, the mitosis inhibitor Wee1 is targeted for degradation during S phase and G2 to allow mitotic entry. Wee1 is an essential tyrosine kinase required for the G2/M transition and S-phase progression. Although several studies have concentrated on Wee1 regulation during mitosis, few have elucidated its degradation during interphase. Our prior studies have demonstrated that Wee1 is degraded via CK1δ dependent phosphorylation during the S and G2/M phases of the cell cycle. Here we demonstrate that GSK3β may work in concert with CK1δ to induce Wee1 destruction during interphase. We generated small molecules that specifically stabilized Wee1. We profiled these compounds against 296 kinases and found that they inhibit GSK3α and GSK3β, suggesting that Wee1 may be targeted for proteolysis by GSK3. Consistent with this notion, known GSK3 inhibitors stabilized Wee1 and GSK3β depletion reduced Wee1 turnover. Given Wee1's central role in cell cycle progression, we predicted that GSK3 inhibitors should limit cell proliferation. Indeed, we demonstrate that GSK3 inhibitors potently inhibited proliferation of the most abundant cell in the mammalian brain, the cerebellar granule cell progenitor (GCP). These studies identify a previously unappreciated role for GSK3β mediated regulation of Wee1 during the cell cycle and in neurogenesis. Furthermore, they suggest that pharmacological inhibition of Wee1 may be therapeutically attractive in some cancers where GSK-3β or Wee1 are dysregulated.     

TRPC1 protein channel is major regulator of epidermal growth factor receptor signaling

TRP channels have been associated with cell proliferation and aggressiveness in several cancers. In particular, TRPC1 regulates cell proliferation and motility, two processes underlying cancer progression. We and others have described the mechanisms of TRPC1-dependent cell migration. However, the involvement of TRPC1 in cell proliferation remains unexplained. In this study, we show that siRNA-mediated TRPC1 depletion in non small cell lung carcinoma cell lines induced G(0)/G(1) cell cycle arrest resulting in dramatic decrease in cell growth. The expression of cyclins D1 and D3 was reduced after TRPC1 knockdown, pointing out the role of TRPC1 in G(1)/S transition. This was associated with a decreased phosphorylation and activation of EGFR and with a subsequent disruption of PI3K/Akt and MAPK downstream pathways. Stimulation of EGFR by its natural ligand, EGF, induced Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry through TRPC1. Ca(2+) entry through TRPC1 conversely activated EGFR, suggesting that TRPC1 is a component of a Ca(2+)-dependent amplification of EGF-dependent cell proliferation.     

Regulation of intracellular pH during the G1/S-phase transition of the neuroblastoma cell cycle

Changes in active K+ and Na+ influx during the cell cycle of neuroblastoma (clone Neuro-2A) have suggested activation of an Na+, H+ exchange system during the G1/S-phase transition. Here we report that pHi, measured by the digitonin null-point method, is constant during G1-phase and the G1/S-phase transition and decreases in early S-phase. In addition pHi is shown to be most sensitive to the diuretic amiloride in the G1/S-phase transition, in agreement with the ion influx data. It is concluded from these data, that pHi is tightly regulated during the early cell cycle phases by the Na+, H+ exchange system, in particular during the G1/S-phase transition.     

Oxygen enhancement ratio as a function of dose and cell cycle phase for radiation-resistant and sensitive CHO cells

There is still controversy over whether the oxygen enhancement ratio (OER) varies as a function of dose and cell cycle phase. In the present study, the OER has been measured as a function of survival level and cell cycle phase using volume flow cell sorting. This method allows both the separation of cells in different stages of the cycle from an asynchronously growing population, and the precise plating of cells for accurate measurements at high survival levels. We have developed a cell suspension gassing and sampling system which maintained an oxygen tension less than 20 ppm throughout a series of sequential radiation doses. For both radiation-resistant cells (CHO-K1) and a radiation-sensitive clone (CHO-xrs6), we could separate relatively pure populations of G1-phase, G1/S-boundary, S-, and G2-phase cells. Each cell line showed a typical age response, with cells at the G1/S-phase boundary being 4 (CHO-K1) to 12 (CHO-xrs6) times more sensitive than cells in the late S phase. For both cell lines, G1-phase cells had an OER of 2.3-2.4, compared to an OER of 2.8-2.9 for S-phase and 2.6-2.7 for G2-phase cells. None of these age fractions showed a dependence of OER on survival level. Asynchronously growing cells or cells at the G1/S-phase boundary had an OER similar to that of G1-phase cells at high survival levels, but the OER increased with decreasing survival level to a value near that of S-phase cells. These results suggest that the decrease in OER at high survival levels for asynchronous cells may be due to differences in the OERs of the inherent cell age subpopulations. For cells in one cell cycle stage, oxygen appears to have a purely dose-modifying effect.     

The Dictyostelium cell cycle and its relationship to differentiation

Abstract The Dictyostelium vegetative cell cycle is characterized by a short mitotic period followed immediately by a short S-phase (less than 30 min) and a long and variable G2 phase. The cell cycle continues during differentiation despite a decrease in cell mass: DNA replication and mitosis occur early in development and also at the tipped aggregate stage. Cells that are in mitosis, S-phase or early G2, when starved differentiate into prestalk cells and cells that are in the middle of G2 differentiate into prespore cells. We postulate that there is a restriction point late in the G2 phase, about 1–2 h before mitosis, where the cells can be arrested either by starvation and the initiation of development, by growing into stationary phase, or by prolonged incubation at low temperature. During development, this block persists to the tipped aggregate stage, where it is specifically released in prespore cells, and these cells then go through one more round of cell division. Genes encoding components of the cell cycle machinery have recently been isolated and attemps to specifically block the cell cycle by reverse genetics to study the effects on differentiation have been initiated.     

Nitrosylation of manganese(II) tetrakis(N-ethylpyridinium-2-yl)porphyrin: a simple and sensitive spectrophotometric assay for nitric oxide.

Reaction between NO(*) and manganese tetrakis(N-ethylpyridinium-2-yl)porphyrin (Mn(III)TE-2-PyP(5+)) was investigated at 25 degrees C. At high excess of NO(*) (1.5 mM) the reaction with the oxidized, air-stable form Mn(III)TE-2-PyP(5+) (5 microM), proceeds very slowly (t(1/2) congruent with 60 min). The presence of excess ascorbate (1 mM) produces the reduced form, Mn(II)TE-2-PyP(4+), which reacts with NO(*) stoichiometrically and in the time of mixing (k congruent with 1 x 10(6) M(-1) s(-1)). The high rate of formation and the stability of the product, Mn(II)TE-2-PyP(NO)(4+) (?Mn(NO)?(6)), make the reaction outcompete the reaction of NO(*) with O(2). Our in vitro measurements show a linear absorbance response upon addition of NO to a PBS, pH 7.4, solution containing an excess of ascorbate over Mn(III)TE-2-PyP(5+). Thus, the observed interactions can be the basis of a convenient and sensitive spectrophotometric assay for NO(*). Also, it may have important implications for the in vivo behavior of Mn(III)TE-2-PyP(5+) which is currently exploited as a possible therapeutic agent for various oxygen-radical related disorders.     

Lymphoblasts already in the DNA synthesis phase of the cell cycle can be reversibly arrested at the R/G transition

Early and late S-phase of the cell cycle are separated by the R-band/G-band (R/G) transition. This corresponds to the time at which R-band synthesis has been completed while G-band synthesis has yet to begin. The aim of this work was to study cell cycle kinetics during S-phase using different blocking agents: mimosine, methotrexate, 5-fluorouracil, 5-fluoro-2'-deoxyuridine and an excess of thymidine. The stage at which these blocking agents arrest the cell cycle and their efficiency at blocking Epstein-Barr virus transformed lymphoblasts at the R/G transition were evaluated using flow cytometric techniques. Mimosine blocked 90% of the cells near the G1/S-phase boundary. Methotrexate, 5-fluoro-2'-deoxyuridine and 5-fluorouracil, and particularly thymidine, let a significant proportion of cells enter S-phase. The cells were released from the arrest state and their progression through early S-phase was monitored by flow cytometry. Before the cells reached the R/G transition, a second agent was added to inhibit cell cycle progression. For example, the use of mimosine followed by thymidine allowed up to 60% of the cells to be blocked at the R/G transition. The arrest of DNA replication at the R/G transition was confirmed by a marked decrease of 5-bromo-2'-deoxyuridine (BrdUrd) incorporation, revealed by using bivariate flow cytometric analysis. The blocking agent was then removed and the cell cohort was released in the presence of BrdUrd so that replication banding analysis could be performed on the harvested mitotic cells. This yielded a mitotic index of approximately 10% and chromosomes showing replication bands. Flow cytometric analysis combined with cytogenetic banding analysis suggested that the R/G transition is an arrest point within the S-phase of the cell cycle and allowed us to conclude that only cells that have already initiated S-phase are blocked at this point. It corresponds to a susceptible site where S-phase can be arrested easily. The R/G transition could also be a regulatory checkpoint within S-phase, a checkpoint that could respond to imbalance in deoxyribonucleotide pools.     

GSK3 inhibitors stabilize Wee1 and reduce cerebellar granule cell progenitor proliferation

Ubiquitin mediated proteolysis is required for transition from one cell cycle phase to another. For instance, the mitosis inhibitor Wee1 is targeted for degradation during S phase and G2 to allow mitotic entry. Wee1 is an essential tyrosine kinase required for the G2/M transition and S-phase progression. Although several studies have concentrated on Wee1 regulation during mitosis, few have elucidated its degradation during interphase. Our prior studies have demonstrated that Wee1 is degraded via CK1δ dependent phosphorylation during the S and G2/M phases of the cell cycle. Here we demonstrate that GSK3β may work in concert with CK1δ to induce Wee1 destruction during interphase. We generated small molecules that specifically stabilized Wee1. We profiled these compounds against 296 kinases and found that they inhibit GSK3α and GSK3β, suggesting that Wee1 may be targeted for proteolysis by GSK3. Consistent with this notion, known GSK3 inhibitors stabilized Wee1 and GSK3β depletion reduced Wee1 turnover. Given Wee1''s central role in cell cycle progression, we predicted that GSK3 inhibitors should limit cell proliferation. Indeed, we demonstrate that GSK3 inhibitors potently inhibited proliferation of the most abundant cell in the mammalian brain, the cerebellar granule cell progenitor (GCP). These studies identify a previously unappreciated role for GSK3β mediated regulation of Wee1 during the cell cycle and in neurogenesis. Furthermore, they suggest that pharmacological inhibition of Wee1 may be therapeutically attractive in some cancers where GSK-3β or Wee1 are dysregulated.     

Initial steps of photosystem II de novo assembly and preloading with manganese take place in biogenesis centers in Synechocystis

In the cyanobacterium Synechocystis sp PCC 6803, early steps in thylakoid membrane (TM) biogenesis are considered to take place in specialized membrane fractions resembling an interface between the plasma membrane (PM) and TM. This region (the PratA-defined membrane) is defined by the presence of the photosystem II (PSII) assembly factor PratA (for processing-associated TPR protein) and the precursor of the D1 protein (pD1). Here, we show that PratA is a Mn(2+) binding protein that contains a high affinity Mn(2+) binding site (K(d) = 73 μM) and that PratA is required for efficient delivery of Mn(2+) to PSII in vivo, as Mn(2+) transport is retarded in pratA(-). Furthermore, ultrastructural analyses of pratA(-) depict changes in membrane organization in comparison to the wild type, especially a semicircle-shaped structure, which appears to connect PM and TM, is lacking in pratA(-). Immunogold labeling located PratA and pD1 to these distinct regions at the cell periphery. Thus, PratA is necessary for efficient delivery of Mn(2+) to PSII, leading to Mn(2+) preloading of PSII in the periplasm. We propose an extended model for the spatial organization of Mn(2+) transport to PSII, which is suggested to take place concomitantly with early steps of PSII assembly in biogenesis centers at the cell periphery.     

Variation in cell-substratum adhesion in relation to cell cycle phases

The quantification of focal adhesion sites offers an assessable method of measuring cell-substrate adhesion. Such measurement can be hindered by intra-sample variation that may be cell cycle derived. A combination of autoradiography and immunolabelling techniques, for scanning electron microscopy (SEM), were utilised simultaneously to identify both S-phase cells and their focal adhesion sites. Electron-energy 'sectioning' of the sample, by varying the accelerating voltage of the electron beam, combined with backscattered electron (BSE) imaging, allowed for S-phase cell identification in one energy 'plane' image and quantitation of immunogold label in another. As a result, it was possible simultaneously to identify S-phase cells and their immunogold-labelled focal adhesions sites on the same cell. The focal adhesion densities were calculated both for identified S-phase cells and the remaining non-S-phase cells present. The results indicated that the cell cycle phase was a significant factor in determining the density of focal adhesions, with non-S-phase cells showing a larger adhesion density than S-phase cells. Focal adhesion morphology was also seen to correspond to cell cycle phase; with 'dot' adhesions being more prevalent on smaller non-S-phase and the mature 'dash' type on larger S-phase cells. This study demonstrated that when quantitation of focal adhesion sites is required, it is necessary to consider the influence of cell cycle phases on any data collected.