Complexity of the mechanisms of initiation and maintenance of DNA damage-induced G2-phase arrest and subsequent G1-phase arrest: TP53-dependent and TP53-independent roles

Through a detailed study of cell cycle progression, protein expression, and kinase activity in gamma-irradiated synchronized cultures of human skin fibroblasts, distinct mechanisms of initiation and maintenance of G2-phase and subsequent G1-phase arrests have been elucidated. Normal and E6-expressing fibroblasts were used to examine the role of TP53 in these processes. While G2 arrest is correlated with decreased cyclin B1/CDC2 kinase activity, the mechanisms associated with initiation and maintenance of the arrest are quite different. Initiation of the transient arrest is TP53-independent and is due to inhibitory phosphorylation of CDC2 at Tyr15. Maintenance of the G2 arrest is dependent on TP53 and is due to decreased levels of cyclin B1 mRNA and a corresponding decline in cyclin B1 protein level. After transiently arresting in G2 phase, normal cells chronically arrest in the subsequent G1 phase while E6-expressing cells continue to cycle. The initiation of this TP53-dependent G1-phase arrest occurs despite the presence of substantial levels of cyclin D1/CDK4 and cyclin E/CDK2 kinase activities, hyperphosphoryated RB, and active E2F1. CDKN1A (also known as p21(WAF1/CIP1)) levels remain elevated during this period. Furthermore, CDKN1A-dependent inhibition of PCNA activity does not appear to be the mechanism for this early G1 arrest. Thus the inhibition of entry of irradiated cells into S phase does not appear to be related to DNA-bound PCNA complexed to CDKN1A. The mechanism of chronic G1 arrest involves the down-regulation of specific proteins with a resultant loss of cyclin E/CDK2 kinase activity.     

Cell-cycle phase-dependence of drug-induced cycle progression delay.

The effect of adriamycin on cell cycle phase progression of CHO cells synchronized into the various phases of the cell cycle by elutriation was investigated by high resolution pulse cytophotometry. Cells treated in all phases of the cell cycle showed delay in their subsequent progression. In addition to the wellknown block of cells in the G2-phase, a delay in passage of cells from G1 to S and a decreased rate of transit through the S-phase were observed. A broadening of the DNA distributions of the treated cells was observed after cell division indicating induction of chromosomal abnormalities.     

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 effects of wortmannin on radiation survival and mutation

Wortmannin, a known radiation sensitizer, has been used in experiments with synchronized cells to compare its effect on radiation survival and mutation induction within the cell cycle. PL61 cells (CHO cells with an inactivated HPRT gene containing a single active copy of a bacterial gpt gene) were synchronized by mitotic selection. Wortmannin administered before gamma irradiation caused a greater sensitization in G(1)-phase cells relative to late S/G(2)-phase cells. Preferential radiosensitization of G(1)-phase cells by wortmannin sets a limit to the proposed use of wortmannin in radiation therapy, since, in contrast to normal tissues, tumors usually have high proportions of S-phase cells. Wortmannin increased mutation frequencies in both G(1)- and S/G(2)-phase cells. Interestingly, relative increases in radiation-induced mutations in G(1) and S/G(2) phases were comparable. The results are discussed in terms of the contributions of different repair modes in the production of mutations.     

Alterations of cell cycle kinetics and vimentin expression in TPA-treated, asynchronous MPC-11 mouse plasmacytoma cells

Vimentin expression throughout the cell cycle has been analyzed at the single-cell level in asynchronously growing MPC-11 cells using multiparameter flow cytometry. We have previously shown that these cells normally lack detectable amounts of intermediate filament proteins. In the presence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), cell proliferation ceases and large quantities of the intermediate filament protein vimentin are synthesized and accumulate in most of the cells. In the present study, the short-term effect of TPA on distribution of cells within the cell cycle was a depletion in early S phase followed by a depletion in mid- and late S phase. In parallel, the G1-phase fraction increased significantly. In addition, a delay in progression through G2/M phase was observed. These data strongly suggest an inhibition of progression of cells through the cell cycle in G1 phase as the primary event on cell cycle kinetics elicited by TPA. Vimentin accumulation could be detected by flow cytometry as early as 2 h after TPA addition; at this time, the percentage of vimentin-positive cells was highest in G2/M phase. Prolonged TPA treatment induced vimentin accumulation in cells of all cell cycle phases. However, even at later times, the G1-phase population consisted of two subpopulations with low and high vimentin content, respectively. The fraction of cells which displayed a higher level of vimentin probably represents those G1-phase cells which previously had undergone cell division in the presence of TPA. Our data indicate that TPA-induced vimentin synthesis is regulated in a cell cycle-dependent manner and is maximally induced in cells which have passed a putative cell cycle restriction point in G1 phase.     

Phosphorylation-dephosphorylation of retinoblastoma protein not necessary for passage through the mammalian cell division cycle

Phosphorylation of the retinoblastoma protein (Rb) during the G1-phase of the mammalian cell division cycle is currently believed to be a controlling element regulating the passage of cells into S-phase. We find, however, that the suspension-grown cell lines U937, L1210, and MOLT-4 contain exclusively hyperphosphorylated Rb. Furthermore, when adherent NIH3T3 cells are grown at very low densities to avoid overgrowth and contact inhibition, they also contain only hyperphosphorylated Rb. NIH3T3 cells exhibit hypophosphorylation when the cells are grown at moderate to high cell densities. We propose that cultures of adherent cells such as NIH3T3, when grown to moderate cell densities, are made up of two populations of cells: (a) cells that are relatively isolated and therefore growing exponentially without contact inhibition, and (b) cells that are growth-inhibited by local cell density or contact inhibition. The common observation in adherent cell lines, that Rb is both hyper- and hypophosphorylated in the G1-phase and only hyperphosphorylated in the S- and G2-phases, is explained by the effects of cell density and contact inhibition. Thus, phosphorylation-dephosphorylation of Rb protein during the G1 phase is not a necessary process during the NIH3T3, L1210, MOLT-4, and U937 division cycles. We propose that phosphorylation-dephosphorylation of Rb is independent of the division cycle and is primarily determined by growth conditions throughout the division cycle.     

Activation of extracellular signal-regulated kinase (ERK) in G2 phase delays mitotic entry through p21CIP1

Extracellular signal-regulated kinase activity is essential for mediating cell cycle progression from G(1) phase to S phase (DNA synthesis). In contrast, the role of extracellular signal-regulated kinase during G(2) phase and mitosis (M phase) is largely undefined. Previous studies have suggested that inhibition of basal extracellular signal-regulated kinase activity delays G(2)- and M-phase progression. In the current investigation, we have examined the consequence of activating the extracellular signal-regulated kinase pathway during G(2) phase on subsequent progression through mitosis. Using synchronized HeLa cells, we show that activation of the extracellular signal-regulated kinase pathway with phorbol 12-myristate 13-acetate or epidermal growth factor during G(2) phase causes a rapid cell cycle arrest in G(2) as measured by flow cytometry, mitotic indices and cyclin B1 expression. This G(2)-phase arrest was reversed by pre-treatment with bisindolylmaleimide or U0126, which are selective inhibitors of protein kinase C proteins or the extracellular signal-regulated kinase activators, MEK1/2, respectively. The extracellular signal-regulated kinase-mediated delay in M-phase entry appeared to involve de novo synthesis of the cyclin-dependent kinase inhibitor, p21(CIP1), during G(2) through a p53-independent mechanism. To establish a function for the increased expression of p21(CIP1) and delayed cell cycle progression, we show that extracellular signal-regulated kinase activation in G(2)-phase cells results in an increased number of cells containing chromosome aberrations characteristic of genomic instability. The presence of chromosome aberrations following extracellular signal-regulated kinase activation during G(2)-phase was further augmented in cells lacking p21(CIP1). These findings suggest that p21(CIP1) mediated inhibition of cell cycle progression during G(2)/M phase protects against inappropriate activation of signalling pathways, which may cause excessive chromosome damage and be detrimental to cell survival.     

Significance of the cell cycle in commitment of murine erythroleukemia cells to erythroid differentiation.

The relationship between differentiation of murine erythroleukemia cells (MEL) induced by DMSO and the cell division cycle has been analyzed. We demonstrate that incubation in the presence of DMSO increases the length of the G1 phase of the cell cycle. A method of synchronization of MEL cells by unit gravity sedimentation has been developed and characterized. Using this method, a series of synchronized cell populations covering the entire cell division cycle can be generated simultaneously. Cells synchronized by this technique were challenged with DMSO and analyzed for kinetics of commitment to the differentiation program. Our results indicate that populations of cells in G1 or G2 at the time of addition of inducer give rise to a greater proportion of committed cells than an unfractionated population, while cells in S phase result in a lower percentage of committed cells than the unfractionated population when cultured in DMSO.     

Lovastatin induces mitotic abnormalities in various cell lines.

We examined the effects of lovastatin, a common anti-atherosclerotic drug and a blocker of the cell cycle, on the process of mitosis. It is known that lovastatin induces an arrest or a retardation of the cell cycle in many cell types not only at the G(1)phase, but also at the G(2)/M transition. After 24-48 h incubation of epithelial PtK(2), T24, HeLa cells and fibroblastic L929 cells in the presence of 1. 0-60.0 microm lovastatin, diverse mitotic perturbations have been observed. The most noteworthy phenomena recorded were prometaphase retardation and chromosome lagging during metaphase and anaphase. After the recovery in lovastatin-free media, the cells continued mitosis without any disturbances. Mevalonic acid prevented the effects of lovastatin. We conclude that the effects were specific for lovastatin-induced inhibition of mevalonic acid synthesis. Immunofluorescence studies with anticentromeric antibodies suggested that one of the possible causes of the lovastatin-induced mitotic disorder could be an interference with the development and function of the centromeres.     

Cell cycle dependent distribution of the proliferation-associated Ki-67 antigen in human embryonic lung cells

The cell cycle-dependent distribution of the proliferation-associated Ki-67 antigen has been evaluated immunocytochemically in L-132 human fetal lung cells. The cells were synchronized and cell cycle phases were determined: G1 = 6.7 h, S = 5.4 h, G2 = 8.5 h and mitosis = 1.3 h. The Ki-67 patterns were strictly correlated with the cell cycle phases. In late G1-phase, Ki-67 antigen was present only in the perinucleolar region. In the S-phase, Ki-67 staining was found homogeneously in the karyoplasm and in the perinucleolar region. G2-phase cells contained a finely granular Ki-67 staining in the karyoplasm with Ki-67-positive specks and perinucleolar staining. In early mitotic cells (pro- and metaphase) an intense perichromosomal Ki-67 staining was observed in addition to a homogeneously stained karyoplasm in prophase, and cytoplasm in metaphase. During ana- and telophase the Ki-67 antigen disappeared rapidly. In resting cells there was no Ki-67 staining.     

Assessment of lovastatin application as tool in probing cytokinin-mediated cell cycle regulation

Lovastatin, a potent inhibitor of the mevalonate pathway, has been used in plant cell cycle studies to eliminate the cytosolic cytokinin biosynthesis. However, several implications can blur the results, as cytokinins may be alternatively formed from isopentenylpyrophosphate produced by the plastid 1-deoxy-xylulose 5-phosphate pathway and because the endogenous cytokinin levels oscillate considerably in the course of a cell cycle. In the work presented here, short- and long-term effects of lovastatin on suspension- cultured Nicotiana tabacum (L.) BY-2 cells were differentiated. The short-term experiments revealed a fast action of lovastatin, resulting in a significantly, though not completely, decreased content of endogenous cytokinins that became visible already after 10 min and was most pronounced after 30 min. But the impact of lovastatin on cell cycle progression depended also on the phase of the cell cycle at which it was administered. Lowering of the cytokinin level during the early S phase, when the endogenous cytokinin levels increased, delayed the S/G2 transition, whereas the same treatment in the late S phase, when the cellular cytokinin concentrations had already started to decrease, promoted it. Incubation periods longer than 48 h resulted in about 50% loss of viable of the cells and also in a reduced capability of division of the survivors. These cells later on resumed cell division. A second treatment with lovastatin of that culture again killed about 50% of the cells, but the surviving cells showed faster re-growth. In conclusion, lovastatin appears as a useful inhibitor of cytokinin biosynthesis in short-term studies, but its use in long-term experiments may create complex effects and therefore requires substantial caution.     

Enhanced sensitivity of G1 arrested human cancer cells suggests a novel therapeutic strategy using a combination of simvastatin and TRAIL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in tumor cells over normal cells. To study the relationship between cell cycle progression and TRAIL-induced apoptosis, SW480 colon cancer and H460 lung cancer cell lines were examined for their sensitivity to TRAIL after arrest in different cell cycle phases. Cells were synchronized in G0/G1, S, and G2/M phase by serum starvation, aphidicolin, or nocodazole treatment, respectively. We found that arrest of cells in G0/G1 phase confers significantly higher susceptibility to TRAIL-induced apoptosis as compared to cells in late G1, S, or G2/M phase. To determine if cell cycle phase could be harnessed for therapeutic gain in the presence of TRAIL, we used the HMG-CoA reductase inhibitor, Simvastatin and lovastatin, to enrich a cancer cell population in G0/G1. Both simvastatin and lovastatin significantly augmented TRAIL-induced apoptosis in tumor cells, but not in normal keratinocytes. The results indicate that TRAIL, in combination with a HMG-CoA reductase inhibitor, may have therapeutic potential in the treatment of human cancer.     

The role of protein metabolism in glucocorticoid-lnduced prolongation of G1 phase in human NHIK 3025 cells

It has previously been found that human NHIK 3025 cells have a glucocortiocoid-sensitive restriction point in mid-G1 phase of the cell cycle. When these cells were synchronized by mitotic selection and exposed to dexamethasone before the restriction point, G1 phase was prolonged whereas the rest of the cell cycle was unperturbed by the hormone. These observations were confirmed by flowcytometric mesurements of synchronized cells in the present study. Cells that received dexamethasone (10^?6 M) just after mitotic selection had a 4 hour prolongation of both G1 and the total cell cycle. However, the general rates of both protein synthesis and protein degradation were found not to be altered by the hormone, i.e., the rate of protein accumulation in dexamethasone exposed cells was equal to that of control cells. Dexamethasone exposed NHIK 3025 cells were found to be larger than control cells at the time of cell division. This is a direct consequence of a prolonged cell cycle duration with no change in general protein metabolism. It thus appears that the dexamethasone-induced prolongation of G1 phase is the result of a steroid-regulated G1 specific process(es) leading toward DNA replication, a process that does not alter general protein accumulation.     

Cell cycle-specific glucocorticoid growth regulation of a human cell line (NHIK 3025)

It has been reported that the human cell line NHIK 3025 has a specific cytoplasmic glucocorticoid receptor. When these cells were exposed to glucocorticoids, the cell cycle time was prolonged. Cells, synchronized by mitotic selection, were subjected to the synthetic glucocorticoid dexamethasone throughout the cell cycle. Only cells exposed in the first half of G1 phase had a lengthened cell cycle time. Most of the prolongation was also located within the G1 phase. The dexamethasone growth inhibition was reversible and could be detected only in the cell cycle where the cells were exposed to the steroid. DNA-histograms of asynchronous cells were recorded by flowcytometry at various times after steroid exposure. These histograms also showed G1 phase sensitivity and G1 phase prolongation after exposure to dexamethasone. Our results thus indicate that these cells have a dexamethasone-sensitive restriction point in mid-G1 phase of the cell cycle.     

Deciliation interferes with cell-cycle progression in Tetrahymena

The impact of ciliary regeneration upon cell-cycle progression of the ciliate Tetrahymena was studied. It was found that cell division ceases during ciliary regeneration, and starts again about 4 h after deciliation. Deciliation of an asynchronously multiplying culture results in a rapid interruption of DNA synthesis, followed by resumption 1 h later. This was shown by pulse-labelling the cells with [3H]thymidine at various times after deciliation. Cytophotometric determinations of the macronuclear DNA content substantiated these observations, since the average DNA content per cell remained constant within the first hour of regeneration, confirming the labelling experiments, after which it rose. At its maximum, the average DNA content was more than doubled as compared with the beginning of the experiment. This indicates that a substantial proportion of the regenerating cells performed two rounds of DNA replication prior to cell division. The massive drop in the average DNA content during the fifth hour after deciliation indicates that the culture becomes partly synchronized for cell division by the deciliation procedure. The division synchrony results from a greater delay of the next cell division when G2 cells are deciliated than occurs in G1 cells. This was shown by deciliating cultures of Tetrahymena thermophila cells in the respective stages of the cell cycle, which had been partly synchronized by elutriator centrifugation. Thus, deciliation followed by ciliary regeneration causes a varying degree of retardation in progression through the cell cycle, being greatest for G2 cells and least for G1 cells.     

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.     

Dibutyryl cyclic AMP arrests the growth of cultivated Cloudman melanoma cells in the late S and G2 phases of the cell cycle.

DBcAMP reversibly arrests cultivated Cloudman melanoma cells in the late S and G2 phases of the cell cycle. This is supported by the measurement of DNA synthesis by autoradiography and measurement of cellular DNA by two methods--the diphenylamine reaction and microspectrophotometry of Feulgen stained cells. We also present evidence that (1) cell division is prevented if DBcAMP is added as late in the cycle as early S phase. (2) The inhibition of cell division does not appear to be caused by products of tyrosine oxidation. (3) The increase in cell size that occurs in the presence of DBcAMP reflects continued synthesis of protein in the absence of cell division.     

Regulation of late G1/S phase transition and APC Cdh1 by reactive oxygen species

Proliferating cells have a higher metabolic rate than quiescent cells. To investigate the role of metabolism in cell cycle progression, we examined cell size, mitochondrial mass, and reactive oxygen species (ROS) levels in highly synchronized cell populations progressing from early G1 to S phase. We found that ROS steadily increased, compared to cell size and mitochondrial mass, through the cell cycle. Since ROS has been shown to influence cell proliferation and transformation, we hypothesized that ROS could contribute to cell cycle progression. Antioxidant treatment of cells induced a late-G1-phase cell cycle arrest characterized by continued cellular growth, active cyclin D-Cdk4/6 and active cyclin E-Cdk2 kinases, and inactive hyperphosphorylated pRb. However, antioxidant-treated cells failed to accumulate cyclin A protein, a requisite step for initiation of DNA synthesis. Further examination revealed that cyclin A continued to be ubiquitinated by the anaphase promoting complex (APC) and to be degraded by the proteasome. This antioxidant arrest could be rescued by overexpression of Emi1, an APC inhibitor. These observations reveal an intrinsic late-G1-phase checkpoint, after transition across the growth factor-dependent G1 restriction point, that links increased steady-state levels of endogenous ROS and cell cycle progression through continued activity of APC in association with Cdh1.     

Strategic cell-cycle regulatory features that provide mammalian cells with tunable G1 length and reversible G1 arrest

Transitions between consecutive phases of the eukaryotic cell cycle are driven by the catalytic activity of selected sets of cyclin-dependent kinases (Cdks). Yet, their occurrence and precise timing is tightly scheduled by a variety of means including Cdk association with inhibitory/adaptor proteins (CKIs). Here we focus on the regulation of G1-phase duration by the end of which cells of multicelled organisms must decide whether to enter S phase or halt, and eventually then, differentiate, senesce or die to obey the homeostatic rules of their host. In mammalian cells, entry in and progression through G1 phase involve sequential phosphorylation and inactivation of the retinoblastoma Rb proteins, first, by cyclin D-Cdk4,6 with the help of CKIs of the Cip/Kip family and, next, by the cyclin E-Cdk2 complexes that are negatively regulated by Cip/Kip proteins. Using a dynamical modeling approach, we show that the very way how the Rb and Cip/Kip regulatory modules interact differentially with cyclin D-Cdk4,6 and cyclin E-Cdk2 provides to mammalian cells a powerful means to achieve an exquisitely-sensitive control of G1-phase duration and fully reversible G1 arrests. Consistently, corruption of either one of these two modules precludes G1 phase elongation and is able to convert G1 arrests from reversible to irreversible. This study unveils fundamental design principles of mammalian G1-phase regulation that are likely to confer to mammalian cells the ability to faithfully control the occurrence and timing of their division process in various conditions.     

Specific G1-S phase cell cycle block by beryllium as demonstrated by cytofluorometric analysis.

Inhibition of cell division by beryllium (Be2+) has been examined in synchronized cultures of a liver-derived cell line (BL9L cells) using cytofluorometric cell cycle analysis. Results show that a selective dose-related block of the G1-pre-S transition is produced, with other periods of the cell cycle appearing relatively insensitive.