Dexamethasone induces irreversible G1 arrest and death of a human lymphoid cell line.

Growth of a human leukemic T-cell line (CEM C7) in 10(-6) M dexamethasone results in inhibition of growth and rapid loss of cell viability after a delay of approximately 18 to 24 hours. Analysis of dexamethasone-treated cells by flow-microfluorometry showed that they were arrested in the G1 phase of the cell cycle. Loss of cell viability began at the same time as G1 accumulation was first detectable, and 20% of all cells were found to be blocked in G1 at this time suggesting that loss of viability and G1 arrest were coincident events. Half-maximal and maximal effects on both viability and G1 arrest after 48 hours in steroid were nearly identical with respect to steroid concentration and corresponded to half-maximal and full occupancy of glucocorticoid specific receptor by hormone, consistent with a glucocorticoid receptor mediated mechanism for both phenomena. Most non-viable cells were arrested in G1, and accumulation of cells in G1 was irreversible; removal of steroid in the presence of colcemid did not result in a decreased fraction of G1 cells. Furthermore, dexamethasone treatment did not protect cells against the effects of 33258 Hoechst-amplified killing of bromodeoxyuridine substituted cells exposed to light. These results show that dexamethasone arrests these leukemic cells in G1 and strongly suggest that dexamethasone-treated cells are killed upon entry into G1.     

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.     

Methylmercury effects on cell cycle kinetics

Methylmercury (MeHg) effects on cell cycle kinetics were investigated to help identify its mechanisms of action. Flow cytometric analysis of normal human fibroblasts grown in vitro in the presence of BrdU allowed quantitation of the proportion of cells in G1, S, G2 and the next G1 phase. This technique provides a rapid and easily performed method of characterizing phase lengths and transition rates for the complete cell cycle. After first exposure to MeHg the cell cycle time was lengthened due to a prolonged G1. At 3 microM MeHg the G1 phase length was 25% longer than the control. The G1/S transition rate was also decreased in a dose-related manner. Confluent cells exposed to MeHg and replated with MeHg respond in the same way as cells which have not been exposed to MeHg before replating. Cells exposed for long times to MeHg lost a detectable G1 effect, and instead showed an increase in the G2 percentage, which was directly related to MeHg concentration and length of exposure. After 8 days at 5 microM MeHg, 45% of the population was in G2. The G2 accumulation was reversible up to 3 days, but at 6 days the cells remained in G2 when the MeHg was removed. Cell counts and viability indicated that there was not a selective loss of cells from the MeHg. MeHg has multiple effects on the cell cycle which include a lengthened G1 and decreased transition probability after short term exposure of cycling cells, and a G2 accumulation after a longer term exposure. There were no detectable S phase effects. It appears that mitosis (the G2 accumulation) and probably synthesis of some macromolecules in G1 (the lengthened G1 and lowered transition probability) are particularly susceptible to MeHg.     

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.     

Glucocorticoid receptor and sequential P53 activation by dexamethasone mediates apoptosis and cell cycle arrest of osteoblastic MC3T3-E1 cells

Glucocorticoids play a pivotal role in the proliferation of osteoblasts, but the underlying mechanism has not been successfully elucidated. In this report, we have investigated the molecular mechanism which elucidates the inhibitory effects of dexamethasone on murine osteoblastic MC3T3-E1 cells. It was found that the inhibitory effects were largely attributed to apoptosis and G1 phase arrest. Both the cell cycle arrest and apoptosis were dependent on glucocorticoid receptor (GR), as they were abolished by GR blocker RU486 pre-treatment and GR interference. G1 phase arrest and apoptosis were accompanied with a p53-dependent up-regulation of p21 and pro-apoptotic genes NOXA and PUMA. We also proved that dexamethasone can't induce apoptosis and cell cycle arrest when p53 was inhibited by p53 RNA interference. These data demonstrate that proliferation of MC3T3-E1 cell was significantly and directly inhibited by dexamethasone treatment via aberrant GR activation and subsequently P53 activation.     

Cell cycle analysis of sodium butyrate and hydroxyurea, inducers of ectopic hormone production in HeLa cells

Sodium butyrate and hydroxyurea, effective inhibitors of DNA synthesis in HeLa cells, cause these cells to produce increased levels of the ectopic glycopeptide hormones human chorionic gonadotropin (hCG), follicle stimulating hormone (FSH), and free alpha chains for these hormones. The objective of this study was an assessment of the role of modulation of cell cycle events in the action of these two chemical agents. A variety of experimental approaches was employed to obtain a clear view of the drugs' effects on cells located initially in all phases of the cell cycle. Cells in early G1, G2, or M phase at time of addition of either inhibitor were not arrested at early time points, but by 48 hours became collected at a location characteristic for each drug, near the G1-S phase boundary. Flow microfluorometry (FMF) and thymidine labeling index revealed that butyrate-treated cells arrested late in G1 phase very close to S phase, while hydroxyurea-blocked cells continued to early S phase. Both inhibitors prevented cells originally in S phase from reaching mitosis. S cells exposed to hydroxyurea were killed by 48 hours, but those growing in 5 mM butyrate progressed to the end of S or G2 phase where they became irreversibly arrested although not removed from the monolayer. Analysis of the cell cycle location and viability of each subpopulation resulting from 48 hour exposure to butyrate or hydroxyurea is important for the study of the function of each cellular subset. Treatment of HeLa cells with lower concentrations of butyrate (1 mM) resulted in slowed yet exponential growth. Fraction labeled mitosis (FLM) analysis shows that this is a result of prolongation of the G1 phase.     

Cell cycle regulation of glucocorticoid receptor function.

Glucocorticoid receptor (GR) nuclear translocation, transactivation and phosphorylation were examined during the cell cycle in mouse L cell fibroblasts. Glucocorticoid-dependent transactivation of the mouse mammary tumor virus promoter was observed in G0 and S phase synchronized L cells, but not in G2 synchronized cells. G2 effects were selective on the glucocorticoid hormone signal transduction pathway, since glucocorticoid but not heavy metal induction of the endogenous Metallothionein-1 gene was also impaired in G2 synchronized cells. GRs that translocate to the nucleus of G2 synchronized cells in response to dexamethasone treatment were not efficiently retained there and redistributed to the cytoplasmic compartment. In contrast, GRs bound by the glucocorticoid antagonist RU486 were efficiently retained within nuclei of G2 synchronized cells. Inefficient nuclear retention was observed for both dexamethasone- and RU486-bound GRs in L cells that actively progress through G2 following release from an S phase arrest. Finally, site-specific alterations in GR phosphorylation were observed in G2 synchronized cells suggesting that cell cycle regulation of specific protein kinases and phosphatases could influence nuclear retention, recycling and transactivation activity of the GR.     

Cell cycle analysis of foreign gene (beta-galactosidase) expression in recombinant mouse cells under control of mouse mammary tumor virus promoter

The cell cycle dependency of foreign gene expression in recombinant mouse L cells was investigated. Two different recombinant mouse L cell lines having the glucocorticoid receptor-encoding gene and the lacZ reporter gene were used in this study. The lacZ gene expression was controlled by the glucocorticoid-inducible mouse mammary tumor virus (MMTV) promoter in both cell lines. In "M4" cells the gr gene was under the control of another MMTV promoter, but in "R2" cells it was under the control of the constitutive Rous sarcoma virus promoter. These normally attachment-grown cells were adapted to suspension culture, and a dual-laser flow cytometer was used to simultaneously determine the DNA and foreign protein (beta-galactosidase) content of single living cells. Expression of beta-galactosidase as a function of cell cycle phase was evaluated for cells in exponential growth without any addition of the glucocorticoid inducer, dexamethasone. Cell cycle positions in the S phase were estimated on the basis of DNA content per cell, and position in the G1 phase was estimated on the basis of cell size as measured by pulse-width time of flight. The results showed that beta-galactosidase synthesis occurred through all cell cycle phases, but the expression rate in the G1 phase was much lower than that in the S and G2/M phases in both cell lines. On the basis of cell size analysis, beta-galactosidase expression in M4 cells (with autoinducible promoter) was found to be higher than that in R2 cells (with inducible promoter) during the G1 phase. (c) 1996 John Wiley & Sons, Inc.     

Cell cycle parameters inAedes albopictus mosquito cells

Summary We have analyzed cell cycle parameters for theAedes albopictus C7-10 mosquito cell line, which has been systematically developed for somatic cell genetics, expression of transfected genes, and synthesis of hormone-inducible proteins. In rapidly cycling cells, we measured a generation time of 10–12 h. The duration of mitosis (M) was ≤1 h, and the DNA synthesis phase (S) required 6 h. UnlikeDrosophila melanogaster Kc cells, in which the G2 gap is substantially longer than G1, in C7-10 cells G1 and G2 each lasted approximately 2h. In these cells, the duration of both S and G2 was independent of the population doubling time, and the increase in population doubling time as cells approached confluency was due to prolongation of G1. When treated with the insect steroid hormone, 20-hydroxyecdysone, C7-10 mosquito cells complete the cycle in progress before undergoing a reversible arrest.     

The cell cycle dependence of thermotolerance. II. CHO cells heated at 45.0 degrees C

This report extends our investigations of the cell cycle dependence of the expression of thermotolerance to include tolerance expressed by Chinese hamster ovary (CHO) cells exposed to 45.0 degrees C hyperthermia. We examined the response of asynchronous cells following exposure at 45.0 degrees C. A maximum in thermotolerance under these conditions was reached approximately 12 hr after a 15-min exposure to 45.0 degrees C hyperthermia and progressively decreased thereafter. Cells were delayed in S and G2 phase for 24 hr, after which time cell growth resumed. We then characterized the response of CHO cell populations synchronized in G1 or early or late S phase. We observed that the expression of tolerance depended on the position of cells in the cell cycle and was modulated by changes in the sensitivity of cells as they progressed through the cell cycle subsequent to the tolerance induction dose. We measured the variation in the sensitivity of these cells to 45.0 degrees C hyperthermia throughout the cell cycle and found substantial changes as cells progressed through S phase. Cells in early S phase were the most sensitive to heat at this temperature, and as these cells progressed through S phase, they became progressively more resistant. In addition, G1 cells were delayed for approximately 15 to 18 hr by a 15-min, 45.0 degrees C heat pulse, whereas S-phase cells were delayed to a lesser extent. The data presented in this report suggest that the induction of thermotolerance is relatively non-cell-cycle specific, but the magnitude of expression of tolerance depends on the position of cells in the cell cycle at the time of the subsequent challenge heat dose.     

Melatonin prevents glucocorticoid inhibition of cell proliferation and toxicity in hippocampal cells by reducing glucocorticoid receptor nuclear translocation

Glucocorticoids are the main product of the adrenal cortex and participate in multiple cell functions as immunosupressors and modulators of neural function. Within the brain, glucocorticoid activity is mediated by high-affinity mineralocorticoid and low-affinity glucocorticoid receptors. Among brain cells, hippocampal cells are rich in glucocorticoid receptors where they regulate excitability and morphology. Also, elevated glucocorticoid levels suppress hippocampal neurogenesis in adults. The pineal neuroindole, melatonin, reduces the affinity of glucocorticoid receptors in rat brain and prevents glucocorticoid-induced apoptosis. Here, the ability of melatonin to prevent glucocorticoid-induced cell death in hippocampal HT22 cells was investigated in the presence of neurotoxins. Results showed that glucocorticoids reduce cellular growth and also enhance sensitivity to neurotoxins. We found a G(1) cell cycle arrest mediated by an increase of cyclin/cyclin-dependent kinase inhibitor p21(WAF1/CIP1) protein after dexamethasone treatment and incremental change in amyloid beta protein and glutamate toxicity. Melatonin prevents glucocorticoids inhibition of cell proliferation and reduces the toxicity caused by glucocorticoids when cells were treated with dexamethasone in combination with neurotoxins. Although, melatonin does not reduce glucocorticoid receptor mRNA or protein levels, it decreases receptor translocation to nuclei in these cells.     

Glucocorticoid receptors and cell cycle progression in human melanoma cell lines

Proliferation of six established human melanoma cell lines was inhibited after treatment for 1 h with a high dose of glucocorticoid. Four of the lines with the capacity of colony formation were used to quantify final plating efficiency. Specific glucocorticoid binding sites in these cell lines ranged from 51,000 to 170,000 sites per cell as measured with a whole-cell assay. Growth inhibition was completely reversible in one cell line, irreversible in another, and partially reversible in two lines. Receptor content per cell correlated with the reduction in final plating efficiency of glucocorticoid-treated cells, suggesting a receptor-mediated event. A more than 90% growth inhibition and a 40% reduction in cell survival in the most sensitive cell line, M-5A, was accompanied by a dual blockage in G1 and G2/M phase that lasted till at least 96 h after treatment with 2.5 microM dexamethasone for 1 h. Evidence is presented of a real arrest of M-5A cells in G1 phase and a markedly retarded progression through G2; the blockage of G1-S transition was immediate and complete. Accumulation of G1 cells was observed in two other cell lines but was inconsistent in the fourth line studied by flow cytometry; in none of the three cell lines was G2/M accumulation observed. Stimulated melanogenesis after glucocorticoid treatment of M-5A and NKI-26 cells suggested differentiation of the cells during glucocorticoid-induced arrest.     

Influence of hormone treatment applied or begun in different phases of the cell cycle on hormonal imprinting in Tetrahymena

Primary exposure to a hormone (hormonal imprinting) alters--in the case of the Tetrahymena increases--cellular response to re-exposure(s) to the same hormone. The intensity of hormonal imprinting depends on the phase of the cell cycle in which the primary exposure has taken place. The effect of imprinting was greater on the cells exposed to the hormone in phase G1 than on those exposed in phase S or G2. The response pattern of the progeny generations corresponded to that of the primarily exposed (imprinted) ancestor cell, irrespective of their own pre-exposure in phase G1, G2 or S of their cycle.     

Changes of mitochondrial respiration,mitochondrial content and cell size after induction of apoptosis in leukemia cells

Mitochondrial damage with release of cytochrome c is implicated in cell death signalling pathways. To examine mitochondrial function in apoptotic cells, we applied high-resolution respirometry to human leukemia cells arrested in the G1- and S-phase by exposure to the glucocorticoid dexamethasone and nucleotide analogue gemcitabine. At 30% apoptosis, opposite effects were observed on respiratory capacity (71% and 131% of controls, respectively). These changes correlated with alterations in cell size, cytosolic, and mitochondrial marker enzymes. Mitochondrial ATP production and membrane potential were maintained in all treatments, as deduced from high respiratory uncoupling control ratios (UCR). Bcl-2 over-expression did not prevent apoptosis after gemcitabine-treatment, but protected dexamethasone-treated cells from apoptosis, without fully preventing the decline of respiration and cell size. These results, therefore, provide conclusive evidence that alterations in respiratory capacity and enzyme activities per cell are mainly caused by opposite changes in cell size, occurring upon cell cycle arrest triggered by dexamethasone and gemcitabine in the early phase of apoptosis.     

Activin A-induced differentiation in K562 cells is associated with a transient hypophosphorylation of RB protein and the concomitant block of cell cycle at G1 phase.

The human erythroleukemic cell line, K562, can be induced to differentiate by the addition of activin A, a newly purified protein belonging to the TGF-beta 1 family. The present studies used flow cytometric cell cycle analysis, indirect immunofluorescence staining of the proliferating cell nuclear antigen (PCNA), and thymidine incorporation assay of cell proliferation to study the effects of activin A on the cell cycle during differentiation in K562 cells. Activin A-treated K562 cells were found to undergo a transient block in cell cycle, temporarily halting progression from G1 to S phase. The latter can be observed after approximately 24 hr of incubation with activin A and then disappears after this early stage of induction of differentiation. Cell cycle kinetics analysis using synchronized K562 cells also confirms that in the presence of activin A, K562 cells progress normally through various phases of cell cycle, except that there is prolongation of the G1 phase between 10 to 24 hr of culture. Furthermore, this transient arrest in G1 is correlated with dephosphorylation of a nucleoprotein, the RB gene product, which occurs within 9-24 hr of incubation with activin A; and phosphorylation of RB protein then develops afterward. In addition, these cell cycle-related events are observed to occur earlier than the accumulation of hemoglobins in K562 cells. It is concluded that transient dephosphorylation of RB protein and prolongation of G1 phase of cell cycle precede and accompany erythroid differentiation caused by activin A and chemical inducers, thus constituting part of the mechanism for induction of differentiation in the erythroleukemia cells.