How the Change from FLM to FACS Affected Our Understanding of the G1-Phase of the Cell Cycle

The frequency of labeled mitoses (FLM) method for analyzing cell-cycle phases necessitates a determination of cell-cycle interdivision times and the absolute lengths of the cell-cycle phases. The change to flow sorting (FACS) analysis, a simpler, less labor intensive, and more rapid method, eliminated determinations of absolute phase times, yielding only percents of cells exhibiting particular DNA contents. Without an interdivision time value, conversion of these fractions into absolute phase lengths is not possible. This change in methodology has led to an alteration in how the cell cycle is viewed. The FLM method allowed the conclusion that G1-phase variability resulted from constancy of S and G2 phase lengths. In contrast, with FACS analysis, slow growing cells exhibiting a large fraction of cells with a G1-phase amount of DNA appeared to be “arrested in G1 phase”. The loss of absolute phase length determinations has therefore led to the proposals of G1-phase arrest, G1-phase controls, restriction points, and G0 phase. It is suggested that these G1-phase controls and phenomena require a critical reevaluation in the light of an alternative cell-cycle model that does not require or postulate such G1-phase controls.     

How the change from FLM to FACS affected our understanding of the G1-phase of the cell cycle

The frequency of labeled mitoses (FLM) method for analyzing cell-cycle phases necessitates a determination of cell-cycle interdivision times and the absolute lengths of the cell-cycle phases. The change to flow sorting (FACS) analysis, a simpler, less labor intensive, and more rapid method, eliminated determinations of absolute phase times, yielding only percents of cells exhibiting particular DMA contents. Without an interdivision time value, conversion of these fractions into absolute phase lengths is not possible. This change in methodology has led to an alteration in how the cell cycle is viewed. The FLM method allowed the conclusion that G1 phase variability resulted from constancy of S and G2 phase lengths. In contrast, with FACS analysis, slow growing cells exhibiting a large fraction of cells with a G1-phase amount of DMA appeared to be "arrested in G1 phase". The loss of absolute phase length determinations has therefore led to the proposals of G1-phase arrest, G1-phase controls, restriction points, and G0 phase. It is suggested that these G1-phase controls and phenomena require a critical reevaluation in the light of an alternative cell-cycle model that does not require or postulate such G1-phase controls.     

Multi parameter in vitro testing of ratjadone using flow cytometry

Ratjadone, isolated from the myxobacterium Sorangium cellulosum, belongs to the family of so-called orphan ligands, which includes leptomycin, callystatin and other compounds. In previous screening tests, ratjadone revealed a growth inhibitory effect against bacteria, yeast and human cancer cells. Following these first results, ratjadone was tested on several human tumour cell lines (Jurkat, HepG2, U87-MG) and, as a control, on a non-tumour cell line (RLC18) for its mode of action. The cell analysis was carried out by flow cytometry. This comprised cell density measurements, live-dead analysis, cell-cycle analysis and detection of apoptosis. First experiments confirmed the growth inhibitory effect on any chosen tumour cell line. Following these results a dose effect relationship was monitored, confirming the high effectiveness of ratjadone against cell growth at nanomolar concentration. Cell cycle analysis has shown that ratjadone intervenes in the cell cycle by arresting the cells in G1-phase. Biological testing of additional ratjadone derivatives with changed configuration and stereochemistry, identified the pharmacophoric site of the molecule.     

Coordinate signaling by integrins and receptor tyrosine kinases in the regulation of G1 phase cell-cycle progression

Cell proliferation is dependent upon the activation of receptor tyrosine kinases and integrins by soluble growth factors and extracellular matrix proteins, respectively. It is now apparent that concerted, rather than individual, signaling by these receptors is the critical feature responsible for cell-cycle progression through G1 phase. ERK (extracellular signal-regulated kinase), Rho GTPases and G1-phase cyclin-dependent kinases are all regulated jointly by growth-factor receptors and integrins. Recent studies have begun to reveal how this regulated signaling in the cytoplasm is linked to activation of the G1-phase cyclin-dependent kinases in the nucleus.     

A generalization of Gompertz law compatible with the Gyllenberg-Webb theory for tumour growth

We present a new extension of Gompertz law for tumour growth and anti-tumour therapy. After discussing its qualitative and analytical properties, we show, in the spirit of [16], that, like the standard Gompertz model, it is fully compatible with the two-population model of Gyllenberg and Webb, formulated in [14] in order to provide a theoretical basis to Gompertz law. Compatibility with the model proposed in [17] is also investigated. Comparisons with some experimental data confirm the practical applicability of the model. Numerical simulations about the method performance are presented.     

Regulation of interkinetic nuclear migration by cell cycle-coupled active and passive mechanisms in the developing brain

A hallmark of neurogenesis in the vertebrate brain is the apical-basal nuclear oscillation in polarized neural progenitor cells. Known as interkinetic nuclear migration (INM), these movements are synchronized with the cell cycle such that nuclei move basally during G1-phase and apically during G2-phase. However, it is unknown how the direction of movement and the cell cycle are tightly coupled. Here, we show that INM proceeds through the cell cycle-dependent linkage of cell-autonomous and non-autonomous mechanisms. During S to G2 progression, the microtubule-associated protein Tpx2 redistributes from the nucleus to the apical process, and promotes nuclear migration during G2-phase by altering microtubule organization. Thus, Tpx2 links cell-cycle progression and autonomous apical nuclear migration. In contrast, in vivo observations of implanted microbeads, acute S-phase arrest of surrounding cells and computational modelling suggest that the basal migration of G1-phase nuclei depends on a displacement effect by G2-phase nuclei migrating apically. Our model for INM explains how the dynamics of neural progenitors harmonize their extensive proliferation with the epithelial architecture in the developing brain.     

Nocodazole does not synchronize cells: implications for cell-cycle control and whole-culture synchronization

It has been predicted that nocodazole-inhibited cells are not synchronized because nocodazole-arrested cells with a G2-phase amount of DNA would not have a narrow cell-size range reflecting the cell size of some specific, presumably G2-phase, cell-cycle age. Size measurements of nocodazole-inhibited cells now fully confirm this prediction. Further, release from nocodazole inhibition does not produce cells that move through the cell cycle mimicking the passage of normal unperturbed cells through the cell cycle. Nocodazole, an archetypal whole-culture synchronization method, can inhibit growth to produce cells with a G2-phase amount of DNA, but such cells are not synchronized. Cells produced by a selective (i.e., non-whole-culture) method not only have a specific DNA content, but also have a narrow size distribution. The current view of cell-cycle control that is based on methods that are not suitable for cell-cycle analysis must therefore be reconsidered when results are based on whole-culture synchronization.This work was supported by the National Science Foundation (grant MCB–0323346) and (in part) by the National Institutes of Health (University of Michigan’s Cancer Center, support grant 5 P30 CA46592). G.I., M.T., and P. B. are associated with the Undergraduate Research Opportunity Program of the University of Michigan, which also supported this research.     

Cell cycle-dependent activation of Ras

Background Ras proteins play an essential role in the transduction of signals from a wide range of cell-surface receptors to the nucleus. These signals may promote cellular proliferation or differentiation, depending on the cell background. It is well established that Ras plays an important role in the transduction of mitogenic signals from activated growth-factor receptors, leading to cell-cycle entry. However, important questions remain as to whether Ras controls signalling events during cell-cycle progression and, if so, at which point in the cell-cycle it is activated.Results To address these questions we have developed a novel, functional assay for the detection of cellular activated Ras. Using this assay, we found that Ras was activated in HeLa cells, following release from mitosis, and in NIH 3T3 fibroblasts, following serum-stimulated cell-cycle entry. In each case, peak Ras activation occurred in mid-G1 phase. Ras activation in HeLa cells at mid-G1 phase was dependent on RNA and protein synthesis and was not associated with tyrosine phosphorylation of Shc proteins and their binding to Grb2. Significantly, activation of Ras and the extracellular-signal regulated (ERK) subgroup of mitogen-activated protein kinases were not temporally correlated during G1-phase progression.Conclusions Activation of Ras during mid-G1 phase appears to differ in many respects from its rapid activation by growth factors, suggesting a novel mechanism of regulation that may be intrinsic to cell-cycle progression. Furthermore, the temporal dissociation between Ras and ERK activation suggests that Ras targets alternate effector pathways during G1-phase progression.     

Cell-cycle progression during the development of Dictyostelium discoideum and its relation to the subsequent cell-sorting in the multicellular structures

Previous studies have shown that the cell-cycle phase at the onset of starvation is a naturally occurring variable that is closely involved in the subsequent sorting and differentiation of cells during Dictyostelium development. Here the cell-cycle progression during the development of D. discoideum Ax-2 cells and its relation to the subsequent cell-sorting were analyzed in detail using synchronized cells and their pulse-labeling by 5'-bromodeoxyuridine (BrdU). Measurements of cell number and nuclearity provided evidence that about 80% of cells progressed their cell-cycle after formation of multicellular structures (mounds). Many cells (T7 cells) starved at mid–late G2-phase (just before the PS-point from which cells initiate development when starved) progressed to the cell-cycle after mound formation. In contrast, a less amount of cells (T1 cells) starved at late G2-phase (just after the PS-point) progressed through the cell-cycle after mound formation. The significance of cell-cycle progression presented here is discussed, with reference to cell differentiation and pattern formation.     

Forecasting the growth of multicell tumour spheroids: implications for the dynamic growth of solid tumours

The growth dynamics of multicell tumour spheroids (MTS) were analysed by means of mathematical techniques derived from signal processing theory. Volume vs. time trajectories of individual spheroids were fitted with the Gompertz growth equation and the residuals (i.e. experimental volume determinations minus calculated values by fitting) were analysed by fast fourier transform and power spectrum. Residuals were not randomly distributed around calculated growth trajectories demonstrating that the Gompertz model partially approximates the growth kinetics of three-dimensional tumour cell aggregates. Power spectra decreased with increasing frequency following a 1/f(delta) power-law. Our findings suggest the existence of a source of 'internal' variability driving the time-evolution of MTS growth. Based on these observations, a new stochastic Gompertzian-like mathematical model was developed which allowed us to forecast the growth of MTS. In this model, white noise is additively superimposed to the trend described by the Gompertz growth equation and integrated to mimic the observed intrinsic variability of MTS growth. A correlation was found between the intensity of the added noise and the particular upper limit of volume size reached by each spheroid within two MTS populations obtained with two different cell lines. The dynamic forces generating the growth variability of three-dimensional tumour cell aggregates also determine the fate of spheroid growth with a strong predictive significance. These findings suggest a new approach to measure tumour growth potential.     

Cytotoxic effects of dexamethasone restricted to noncycling,early G1-phase cells of L1210 leukemia

The cytostatic and cytolytic effects of dexamethasone were studied as functions of cell cycle position in mouse L1210 leukemia cells. To this end, the cells were separated according to size by sedimentation at unit gravity in a specially designed sedimentation chamber. The fractions were analyzed by radioautography and flow cytophotometry. The size-distributions obtained by 1g sedimentation coincided with cell-cycle age distribution. With increasing fraction number, samples highly enriched in G1, S, and G2/M cells, respectively were obtained: the smallest cells being in early G1 and the largest in mitosis. In the presence of dexamethasone (10^?6-10^?5 M), growth slowed down after a few cell cycles and the cells accumulated in early G1 phase. Lytic cell kill by continued exposure to the drug was confined to the fractions containing the small, early G1-phase cells. These fractions were also enriched in noncycling cells that were not labeled by prolonged exposure to ³H-thymidine. After removal of dexamethasone, the cells in S and G2/M phase completed cell cycle traverse but were retarded again in the G1 and early S phase of the next division cycle. The data suggest a memory effect for previous drug exposure. It is concluded that the cytostatic and cytolytic effects of dexamethasone are separate, though not unrelated events. Cytolysis is confined to the noncycling cells that in untreated populations can exit from the dividing compartment during a transitional phase of about 60 minutes subsequent to mitotic division. The cytostatic effects potentiate cytolysis by accumulating the cells in the early G1 phase and thus increasing the probability of their transit to the G0 compartment, sensitive for drug-mediated cytolysis.     

Early growth response (EGR)-1 is required for timely cell-cycle entry and progression in hepatocytes after acute carbon tetrachloride exposure in mice

Cell-cycle induction in hepatocytes protects from prolonged tissue damage after toxic liver injury. Early growth response (Egr)-1(-/-) mice exhibit increased liver injury after carbon tetrachloride (CCl(4)) exposure and reduced TNF-α production. Because TNF-α is required for prompt cell-cycle induction after liver injury, here, we tested the hypothesis that Egr-1 is required for timely hepatocyte entry into the cell cycle after CCl(4)-induced liver injury. Acute liver injury was induced by a single injection of CCl(4). Assays were employed to assess indices of the cell cycle in liver after CCl(4) exposure. Bromodeoxyuridine incorporation peaked in wild-type mice at 48 h after CCl(4) but was reduced by 80% in Egr-1(-/-) mice. Proliferating-cell nuclear-antigen immunohistochemistry revealed blocks in cell-cycle entry and progression to DNA synthesis in Egr-1-deficient mice 48 h after CCl(4). Cyclin D, important for G0/G1 progression, was reduced at baseline and 36 h after CCl(4). Cyclin E1, required for G1/S-phase transition, was reduced in Egr-1(-/-) mice 24 and 48 h after CCl(4) exposure and was associated with reduced phosphorylation of the retinoblastoma protein. Proliferation in Egr-1(-/-) mice was delayed, rather than blocked, because indices of cell-cycle progression were restored 72 h after CCl(4) exposure. We concluded that Egr-1 was required for prompt cell-cycle entry (G0- to G1-phase) and G1/S-phase transition after toxic liver injury. These data support the hypothesis that Egr-1 provides hepatoprotection in the CCl(4)-injured liver, attributable, in part, to timely cell-cycle induction and progression.     

A two-parameter flow cytometry protocol for the detection and characterization of the clastogenic, cytostatic and cytotoxic activities of chemicals

Cultured, freshly-isolated rat fibroblasts were exposed in vitro to vincristine sulphate (VC), amethopterin (AM), bleomycin (BL), benomyl (BE) and practolol (PR). Cells treated for 5 h were subjected 24 h later to a two-parameter (DNA/protein) flow cytometry analysis. The fluorochromes used were sulphorhodamin 101 and DAPI. From DNA and protein histograms, alterations in cell-cycle kinetics, variations in the amount of DNA in individual G1-phase cells and the enhancement of or increased variation in the protein content of the exposed cells were determined. Each of the 5 chemicals induced a specific dose-dependent pattern of changes in the DNA and protein histograms. DNA dispersion was enhanced with VC, AM, BL and BE but not with PR. The cell cycle was blocked in the G2 phase with VC, at early S phase with amethopterin and, depending on the dose, at the G1 or G2 phase with bleomycin or at the S phase or G2 phase with benomyl. Practolol inhibited cells slightly in the S phase at the highest exposure level. Protein analysis allows cytotoxic activity (loss of proteins) or induced unbalanced growth (protein accumulation) of test compounds to be recognized. The results obtained imply that the proposed two-parameter DNA/protein analysis by flow cytometry is a suitable method for prospective testing of chemicals for their induction of structural or numerical chromosome aberrations. Simultaneously, a broad range of cytotoxic, cytostatic and cell-cycle perturbing activities of the test agents can be recognized.     

Sanguinarine-induced G1-phase arrest of the cell cycle results from increased p27KIP1 expression mediated via activation of the Ras/ERK signaling pathway in vascular smooth muscle cells

The present study identified a novel mechanism for the effects of sanguinarine in vascular smooth muscle cells (VSMC). Sanguinarine treatment of VSMC resulted in significant growth inhibition as a result of G₁-phase cell-cycle arrest mediated by induction of p27KIP1 expression, and resulted in a down-regulation of the expression of cyclins and CDKs in VSMC. Moreover, sanguinarine-induced inhibition of cell growth appeared to be linked to activation of Ras/ERK through p27KIP1-mediated G₁-phase cell-cycle arrest. Overall, the unexpected effects of sanguinarine treatment in VSMC provide a theoretical basis for clinical use of therapeutic agents in the treatment of atherosclerosis.     

Distinction of G0 cells from senescent cells in cultures of non-cycling human fetal lung fibroblasts by anti-MAP-1 monoclonal antibody staining

On staining with a monoclonal antibody raised against microtubule-associated protein-1 (MAP-1), dot-like structures were seen in the nuclei of interphase cells, but not in those of non-cycling G0-arrested cells. Dots were also not seen in the nuclei of non-cycling senescent human cells (IMR-90). A SV40-DNA-transformed subline of IMR-90 with a limited growth potential showed progressive decrease of cells with nuclei containing dots in the final stage of their lifespan. The dots appeared in G0-arrested IMR-90 cells when these cells were incubated in medium of high osmotic pressure for 3 min. In contrast, no dots appeared in senescent cells or X-ray-irradiated young cells when they were incubated in medium of high osmotic pressure. Thus irreversibly non-cycling cells could be distinguished from G0-phase cells on the level of whole cultures. The results suggest that senescent cells lose their division potential by entering an irreversible cell-cycle stage differing from G0.     

Complementation by a cloned human ubiquitin-activating enzyme E1 of the S-phase-arrested mouse FM3A cell mutant with thermolabile E1.

A temperature-sensitive growth mutant tsFS20 isolated from mouse FM3A cells was identified as a mutant with thermolabile ubiquitin-activating enzyme E1 by transfection with a full-length cDNA encoding the human E1 enzyme and cell-cell hybridization with an authentic E1 mutant ts85 previously isolated from FM3A cells. The resulting transformants produced thermoresistant E1 activity. Upon shift-up of temperature, asynchronously growing tsFS20 cells showed multiple points of cell-cycle arrest. At the nonpermissive temperature, tsFS20 cells that had been synchronized at the G1-S-phase progressed and accumulated in the mid-S-phase, as evidenced by the absence of G2-specific cdc2 kinase activity, while ts85 mutant cells, the widely used E1 mutant, reached the G2-phase and were arrested. Thus, the E1 mutation seemed to be involved in progression in the S-phase as well as in the G2-phase in the cell cycle. Degradation of short-lived abnormal proteins in tsFS20 cells was decreased to about 50% at the nonpermissive temperature, while the block was fully restored to the wild-type level in the transformant cells. Relevance of the unusually high incidence of the temperature-sensitive E1 mutation was discussed in terms of the E1 as a determinant of heat tolerance of cells.     

On-line gas analysis in animal cell cultivation: I. Control of dissolved oxygen and pH

To monitor gas reaction rates in animal cell culture at constant dissolved oxygen concentration (DO) and constant pH it was necessary to develop improved control methods. Decoupling of both controllrs was obtained by manipulation of molar fractions of oxygen and carbon dioxide in the gas phase. Two pairs of DO and pH controllers were designed and tested both in simulation and exprimental runs. The first controller pair was developed for headspace aeration only, whereas the second controller pair was designed for bubble aeration using a microsparger and flushing the headspace with helium. pH was controlled by a conventional discrete PID controller in its velocity form. For DO control two linear state space feedback controllers with parameter adaptation were established. In these controllers the oxygen uptake rate (OUR) was considered as a disturbance and was not included in the mathematical model. The feedback gain adaptation was based on the difference between the actual molar fraction of oxygen at time step n and the initial molar fraction. This difference is related to OUR and was used to increase or decrease the state feedback controller gain (k and k(1), respectively) in a slow manner. With these controllers it was possible to get an excellent online estimate of OUR. In the case of bubble aeration a simple gas phase mass balance was sufficient, whereas during the headspace aeration a liquid phase balance was required. It has been shown that determination of OUR using gas balance requires a significantly better controller performance compared to just keeping DO and pH within reasonable limits. (c) 1995 John Wiley & Sons, Inc.     

Cell cycle-dependent expression of Kv1.5 is involved in myoblast proliferation

Voltage-dependent K(+) channels (Kv) are involved in the proliferation of many types of cells, but the mechanisms by which their activity is related to cell growth remain unclear. Kv antagonists inhibit the proliferation of mammalian cells, which is of physiological relevance in skeletal muscle. Although myofibres are terminally differentiated, some resident myoblasts may re-enter the cell cycle and proliferate. Here we report that the expression of Kv1.5 is cell-cycle dependent during myoblast proliferation. In addition to Kv1.5 other Kv, such as Kv1.3, are also up-regulated. However, pharmacological evidence mainly implicates Kv1.5 in myoblast growth. Thus, the presence of S0100176, a Kv antagonist, but not margatoxin and dendrotoxin, led to cell cycle arrest during the G(1)-phase. The use of selective cell cycle blockers showed that Kv1.5 was transiently accumulated during the early G(1)-phase. Furthermore, while myoblasts treated with S0100176 expressed low levels of cyclin A and D(1), the expression of p21(cip-1) and p27(kip1), two cyclin-dependent kinase inhibitors, increased. Our results indicate that the cell cycle-dependent expression of Kv1.5 is involved in skeletal muscle cell proliferation.