CELL CYCLE KINETICS OF ENDOREDUPLICATION IN GAMMA-IRRADIATED ROOT MERISTEMS OF PISUM SATIVUM

Label and mitotic indices and microspectrophotometry of unlabeled interphase cells were used to measure the proportion of root meristem cells of Pisum sativum in each cell cycle stage after exposure to protracted gamma irradiation. Three seedling types were investigated: 1) intact seedlings, 2) seedlings with cotyledons detached and treated with lanolin paste applied to the area of cotyledon excision, and 3) seedlings with detached cotyledons and treated with a G2 Factor applied to the area of cotyledon excision in lanolin paste. In intact seedling meristems, predominant cell arrest occurred with a 4C amount of DNA while 0.30 of the cells underwent endoreduplication to arrest with an 8C amount of DNA. Only 0.07 cells arrested with a 2C amount of DNA. Polyploid cells were produced several days after the start of irradiation and were derived from a diploid cell population. In seedlings exposed to lanolin only, without cotyledons, most cells arrested with a 2C amount of DNA with no polyploid cells. In seedlings exposed to a G2 Factor in lanolin after cotyledon excision, most cells arrested with a 4C amount of DNA but no cells underwent endoreduplication. These experimental results suggest that the G2 Factor derived from cotyledons of Pisum sativum was necessary for predominant cell arrest in G2 but alone was not sufficient for the polyploidization step.     

The dose dependent effect of cyclic AMP on ribonucleotide reductase in mitogen stimulated mononuclear cells

Cyclic adenosine monophosphate arrests proliferating T lymphocytes in the G1 phase of the cell cycle. Here we demonstrate that exogenous and endogenous elevations in cyclic AMP concentration result in diminished mitogen stimulation, cell cycle arrest, and decreased ribonucleotide reductase messenger RNA concentrations in peripheral blood mononuclear cells. At lower concentrations (less than 1mM) of dibutyryl cyclic AMP that do not generate cell cycle arrest there is inhibition of ribonucleotide reductase activity without decreased messenger RNA concentration for the M2 subunit of ribonucleotide reductase. However, at higher concentrations of dibutyryl cyclic AMP there is G1 cell cycle arrest and reduced M2 specific messenger RNA concentration. Thus, cyclic AMP inhibition of lymphocyte activation may occur by different mechanisms that are dose dependent.     

视黄酸对胃癌细胞周期的调控

视黄酸(RA)能够抑制许多类型癌细胞生长、诱导细胞凋亡和调节细胞周期。本文研究了全反式视黄酸(ATRA)对人胃癌细胞的作用机理。结果表明,ATRA通过诱导细胞滞留在G_0/G_1期而显著抑制胃癌细胞生长,但ATRA不能诱导胃癌细胞凋亡;ATRA调控细胞周期与c-myc、磷酸化Rb水平的下调和p21~(WAF1/CIP1)、p53水平的上调有关,而cyclinD_1和CDK_4水平没有明显变化。在RA抗性细胞中,ATRA不能调节这些基因表达。结果证实,ATRA对胃癌细胞生长抑制与其诱导细胞滞留在G_0/G_1期有关,而与细胞凋亡的诱导无关,许多重要的、与周期相关的分子,包括cmyc、p21~(WAF1/CIP1、p53和Rb等参与细胞周期的调控。     

M2 subunit of ribonucleotide reductase is a target of cyclic AMP-dependent protein kinase

Cyclic AMP arrests T lymphocytes in the G1 phase of the cell cycle, and prolonged exposure results in cytolysis. Both of these effects require cyclic AMP-dependent protein kinase. We recently observed that some S49 mouse T lymphoma cell lines selected for hydroxyurea resistance were not arrested in G1 by cyclic AMP. Further analysis revealed that these cell lines were cyclic AMP-dependent protein kinase deficient, and conversely, other cyclic AMP-dependent protein kinase deficient cell lines not selected for hydroxyurea resistance were two- to threefold more hydroxyurea resistant. However, hydroxyurea is a specific inhibitor of ribonucleotide reductase and does not inhibit this kinase. We subsequently showed that cyclic AMP-dependent protein kinase will phosphorylate the M2 but not the M1 subunit of ribonucleotide reductase in vitro, and this phosphorylation will diminish CDP reductase activity. In vivo phosphorylation of M2 occurred under conditions similar to those that generate cell cycle arrest. We conclude that the M2 subunit of ribonucleotide reductase can be a target of cyclic AMP-dependent protein kinase. The phosphorylated enzyme has diminished activity, and this may play a role in cyclic AMP-induced lymphocyte cell cycle arrest.     

The control of growth of mouse mastocytoma Cells by N6,O2′-dibutyryladenosine cyclic 3′,5′-monophosphate

Summary Addition of N⁶,O^2′-Dibutyryladenosine cyclic 3′,5′ monophosphate (DB cyclic AMP) plus theophylline or transfer to medium containing 0.2% serum slowed the growth of cultured mouse mastocytoma cells and eventually arrested their growth in G1 phase. Examination of the properties of cells arrested by either procedure suggested that the drugs arrested cells in G1 phase 1.5–2 h after the point of low serum arrest. Cycloheximide prevented the recovery of cell growth after low serum or drug-induced arrest demonstrating that protein synthesis was necessary to pass either growth restriction point. Cordycepin also prevented drug-arrested cells from progressing into cycle indicating a requirement for RNA synthesis to overcome the drug-induced growth arrest. Evidence is also presented that DB cyclic AMP prevented the cells receiving a pulse of calcium necessary to proceed past the DB cyclic AMP-sensitive growth restriction point. It is suggested that high cyclic AMP levels prevent mastocytoma cells from receiving a surge of calcium in G1 phase that is necessary if the cells are to proceed to S phase and eventually divide.     

STIMULATION OF BRAIN SEROTONIN SYNTHESIS BY DIBUTYRYL-CYCLIC AMP IN RATS

Cyclic AMP and dibutyryl-cyclic AMP, a derivative of cyclic AMP resistant to phosphodiesterase inactivation, were injected into the lateral ventricles of rats. These nucleotides did not change the level of brain 5-HT but increased the brain level of its principal metabolite, 5-hydroxyindoleacetic acid. Cyclic AMP was less potent than dibutyryl-cyclic AMP. Butyrate and 5′-AMP were inactive. The effect of dibutyryl cyclic AMP on 5-HT metabolism was studied both in vivo and in vitro. The rate of synthesis of 5-HT was measured by the rate of accumulation of 5-hydroxyindoleacetic acid after the transport of this acid out of the brain was blocked with probenecid. The rate of synthesis of brain 5-HT increased from 0-38 μg/g/h in control rats to 0-65 μg/g/h after dibutyryl-cyclic AMP. In addition cyclic AMP and dibutyryl-cyclic AMP markedly increased brain tryptophan, while AMP was inactive. Since brain tryptophan hydroxylase has a K_m for its substrate that is much higher than the concentrations of tryptophan normally present in the brain, it is likely that the increase in the rate of synthesis of brain 5-HT is secondary to the cyclic AMP induced increase in the levels of brain tryptophan. In vitro studies revealed that dibutyryl-cyclic AMP increased the uptake of radioactive labelled tryptophan into slices of rat brain stem and the formation of 5-HT and 5-hydroxyindoleacetic acid.     

On induction of cell differentiation by cyclic AMP pulses in Dictyostelium discoideum

Repeated pulses of cyclic AMP, applied at intervals of 5 min, efficiently induced differentiation in cells of agip 53, a morphogenetic mutant of Dictyostelium discoideum, strain Ax-2. In contrast, pulses applied at intervals of 2 min did not induce cell differentiation. To analyze this phenomenon the hydrolysis of cyclic AMP between the pulses as well as the effect of the pulses on the intracellular concentration of cyclic GMP were investigated. Experiments performed in the presence of added cyclic AMP phosphodiesterase revealed that incomplete hydrolysis of cyclic AMP was not the reason for the inefficiency of the pulses applied with a 2-min rhythm. Cyclic AMP pulses applied at intervals of 2 min induced discrete increases of the cyclic GMP concentration. Limited time resolution at the level of cyclic GMP cannot account for the inefficiency of the 2-min pulses.Based on material presented at the Symposium Intercellular Communication Stuttgart, September 16–17, 1982     

Auxin-induced growth of tuber tissue of Jerusalem artichoke VIII. Role of cyclic AMP in the action of auxin, cytokinin and gibberellic acid

Cyclic AMP showed no growth-promoting effect when given aloneto unaged slices excised from Jerusalem artichoke tubers. Butit synergistically enhanced cell expansion when given togetherwith an auxin, 2,4-dichlorophenoxyacetic acid. Growth responsesof aged slices to cyclic AMP were much smaller than those ofunaged slices. Cyclic AMP was substantially effective when sliceswere aged in the presence of cyclic AMP, then were transferredto a growth solution containing auxin. Interactions betweencyclic AMP and gibberellic acid or kinetin were additive inpromoting auxininduced cell expansion. Both caffeine and theophylline,inhibitors of phosphodiesterase, enhanced the stimulating effectof applied cyclic AMP on auxin-induced cell expansion. But theydid not enhance the promoting effect of gibberellic acid orkinetin on auxininduced cell expansion. These results suggestthat cyclic AMP did not act as a second messenger for any auxin,gibberellic acid and cytokinin in the promotion of cell expansionin this tissue. (Received September 27, 1972; )     

CELL CYCLE AND CELL MORTALITY OF ALEXANDRIUM MINUTUM (DINOPHYCEAE) UNDER SMALL‐SCALE TURBULENCE CONDITIONS1

Decreased net population growth rates and cellular abundances have been observed in dinoflagellate species exposed to small‐scale turbulence. Here, we investigated whether these effects were caused by alterations in the cell cycle and/or by cell mortality and, in turn, whether these two mechanisms depended on the duration of exposure to turbulence. The study was conducted on the toxic dinoflagellate Alexandrium minutum Halim, with the same experimental design and setup used in previous studies to allow direct comparison among results. A combination of microscopy and Coulter Counter measurements allowed us to detect cell mortality, based on the biovolume of broken cells and thecae. The turbulence applied during the exponential growth phase caused an immediate transitory arrest in the G2/M phase, but significant mortality did not occur. This finding suggests that high intensities of small‐scale turbulence can alter the cell division, likely affecting the correct chromosome segregation during the dinomitosis. When shaking persisted for >4 d, mortality signals and presence of anomalously swollen cells appeared, hinting at the activation of mechanisms that induce programmed cell death. Our study suggests that the sensitivity of dinoflagellates to turbulence may drive these organisms to find the most favorable (calm) conditions to complete their division cycle.     

COMPARISON OF LEVELS OF ADENOSINE 3'',5''-MONOPHOSPHATE IN HIGHLY PURIFIED AND MIXED PRIMARY CULTURES OF NEURONS AND NON-NEURONAL CELLS FROM EMBRYONIC CHICK SYMPATHETIC GANGLIA

Primary cultures containing ≥99% neurons, ≥99% non-neuronal cells (glia), or both cell types were prepared from the sympathetic ganglia of 12-day chick embryos. Levels of cyclic AMP in the non-neuronal cells (～14 pmol/mg protein) were approximately 3-fold higher than levels in the neurons (～4 pmol/mg protein). Mixed cultures had concentrations of cyclic AMP which fell between the values measured for pure neuronal and pure non-neuronal cultures. The measured cyclic AMP values of mixed cultures were indistinguishable from values predicted by summing the expected contributions of the neurons and non-neuronal cells. Thus, contact between the neurons and non-neuronal cells in these mixed cultures did not appear to alter the level of cyclic AMP in either cell type. Neuronal-glial interactions, such as the specific neuronal stimulation of non-neuronal cell proliferation, occurred independently of any changes in the level of cyclic AMP in the mixed cultures. Cell density was varied in both pure and mixed cultures, and both cyclic AMP concentrations and amounts of [³H]thymidine incorporation into DNA were measured. The cyclic AMP content of the non-neuronal cells varied inversely with cell density. [³H]Thymidine incorporation was independent of cell density in both neuronal and non-neuronal cultures. Parallel density-dependent decreases in cyclic AMP concentration and [³H]thymidine incorporation were observed in mixed cultures as cell density was increased. The data suggest that there is no relationship between changes in rate of non-neuronal cell proliferation and cyclic AMP levels in these cultures.     

Periodic stimuli are more successful than randomly spaced ones for inducing development inDictyostelium discoideum

Aggregation in the cellular slime moldDictyostelium discoideum is due to chemotaxis. The chemoattractant, cyclic AMP, is synthesised and released periodically by the cells. Externally applied periodic pulses of cyclic AMP can also induce differentiation in this organism. The present work examines the role of periodicityper se in cyclic AMP-mediated stimulation of cell differentiation. For this purpose we use Agip53, aDictyostelium mutant which does not develop beyond the vegetative state but can be made to aggregate and differentiate by reiterated applications of cyclic AMP. Importantly, Agip53 cells do not make or release any cyclic AMP themselves even in response to an increase in extracellular cyclic AMP. A comparison of the relative efficiencies of periodic and aperiodic stimulation shows that whereas the two patterns of stimulation are equally effective in inducing the formation of EDTA-stable cell contacts, periodic stimuli are significantly superior for inducing terminal differentiation. This suggests that there must be molecular pathways which can only function when stimulation occurs at regular intervals.     

Deoxyribonucleotide metabolism and cyclic AMP resistance in hydroxyurea-resistant S49 T-lymphoma cells

We investigated the cell cycle regulation of deoxyribonucleoside triphosphate (dNTP) metabolism in hydroxyurea-resistant (HYUR) murine S49 T-lymphoma cell lines. Cell lines 10- to 40-fold more hydroxyurea-resistant were selected in a stepwise manner. These HYUR cells exhibited increased CDP reductase activity (5- to 8-fold) and increased dNTP pools (up to 5-fold) that appeared to result from increased activity of the M2 subunit (binding site of hydroxyurea) of ribonucleotide reductase. These characteristics remained stable when the cells were grown in the absence of hydroxyurea for up to 2 years. In both wild type and hydroxyurea-resistant cell populations synchronized by elutriation, dCTP and dTTP pools increased in S phase, whereas dATP and dGTP pools generally remained the same or decreased, suggesting that allosteric effector mechanisms were operating to regulate pool sizes. Additionally, CDP reductase activity measured in permeabilized cells increased in S phase in both wild type and hydroxyurea-resistant cells, suggesting a nonallosteric mechanism of increased ribonucleotide reductase activity during periods of active DNA synthesis. While wild type S49 cells could be arrested in the G1 phase of the cell cycle by dibutyryl cyclic AMP, hydroxyurea-resistant cell lines could not be arrested in the G1 phase by exogenous cyclic AMP or agents that elevate the concentration of endogenous cyclic AMP. These data suggest that cyclic AMP-generated G1 arrest in S49 cells might be mediated by the M2 subunit of ribonucleotide reductase.     

Effects of Cyclic AMP on Components of the Cell Cycle Machinery Regulating DNA Synthesis in Cultured Astrocytes

Cyclic AMP is a second messenger for various hormones that inhibits cell multiplication and DNA synthesis in cultured astrocytes. We examined the effects of increasing intracellular cyclic AMP on the catalytic (cdks) and regulatory (cyclins and ckis) components of cyclin-dependent protein kinases, which regulate progression of the cell cycle before completion of DNA synthesis, in primary cultured astrocytes and in an astrocytic cell line C.LT.T.1.1. The amount of cdk4 changed little during the cell cycle and was not affected by cyclic AMP. There was little cdk1 and cdk2 in quiescent cells, and their expression increased during the G1-S phases. Cyclic AMP strongly inhibited cdk1 and cdk2 expression. Transforming growth factor beta also inhibited cdk1 expression in primary astrocytes. Cyclic AMP did not affect the two ckis p27KIP1 and p21CIP1. There was little cyclin D1 in quiescent cells, but it increased during the G1 phase and was reduced by cyclic AMP. Cyclin E increased during the G1-S phases and was not affected by cyclic AMP in primary astrocytes. The amount of cyclin A was low in quiescent cells and increased during the G1-S phases. Expression of its mRNA and protein was inhibited by cyclic AMP. The protein kinase activities associated with complexes of cyclins and cdks were increased by growth factors and prevented by cyclic AMP. We conclude that cyclic AMP inhibits progression of the cell cycle in astrocytes at least by preventing the expression of the regulatory subunits, cyclins D1 and A, and catalytic subunits, cdk1 and cdk2, of cyclin-regulated protein kinases. Key Words: Cyclin-dependent protein kinases-Glial cells-Cyclic AMP.     

Regulation of purine biosynthesis in G1 phase-arrested mammalian cells

The effects of G1 phase growth arrest on purine biosynthesis were studied in cultured S49 T lymphoma cells. Incubations of wildtype S49 cells for 18 hr with dibutyryl cyclic AMP or forskolin, two agents which induced G1 arrest, reduced the rates of purine biosynthesis by 95%. Time course and concentration dependence studies indicated that the decrease in rates of purine biosynthesis correlated with the extent of G1 phase arrest. Similar studies with somatic cell mutants deficient in some component of cyclic AMP action or metabolism indicated that the depression in purine synthetic rates required G1 arrest and did not result from cell death. Rates of RNA and DNA synthesis were also markedly diminished in the growth arrested cells. Measurements of purine rates in the presence of azaserine indicated that the block in purine biosynthesis was prior to the formation of phosphoribosylformylglycinamide. Additionally, the activities of adenylosuccinate synthetase and IMP dehydrogenase were diminished in G1 arrested cells. The levels of all controlling enzymes, substrates, and cofactors, however, were not diminished in G1 arrested cells. Despite diminished rates of purine biosynthesis, the amounts of intracellular nucleotides in G1 cells were equivalent to those in exponentially growing cells. However, the concentrations of intracellular nucleotides were 30-50% higher in the growth arrested cells. These results suggested that perturbations in the consumption of nucleotides via inhibition of nucleic acid synthesis have profound effects on the purine pathway and indicated the importance of feedback inhibition by nucleotides in the regulation of purine synthesis in situ.     

Evidence that a glucose-mediated rise in cyclic AMP triggers germination ofPilobolus longipes spores

Spores ofPilobolus longipes incubated in phosphate buffer were activated within 5–10 min following the addition of either glucose or 6-deoxyglucose. Cyclic AMP content increased in response to glucose or 6-deoxyglucose, and the increase consistently preceded spore activation. Dibutyryl cyclic AMP also caused activation. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) did not cause activation, but, when added to spores with a suboptimal level of 6-deoxyglucose, it amplified the signal to produce a large increase in activation. IBMX increased intracellular cyclic AMP levels when it was applied with 6-deoxyglucose, but had no effect when it was applied alone. Phosphodiesterase activities in cell extracts from dormant and activated spores were not significantly different. These results indicate that the rise in cyclic AMP that follows exposure to glucose may play an important role in triggering spore germination.     

Effect of dibutyryl cyclic AMP on the restoration of contact inhibition in tumor cells and its relationship to cell density and the cell cycle

KB cell cultures exposed to 10⁻⁴ M dibutyryl cyclic AMP were significantly inhibited and exhibited contact inhibition of growth at cell densities of 8 × 10⁴/cm² irrespective of the initial plating density. Control cultures reached densities of 2.5 × 10⁵/cm². Inhibition of growth did not occur in KB cells when the density was below 1 × 10⁴ cells/cm². When dibutyryl cyclic AMP was removed from KB cells in the contact-inhibited state, growth resumed with DNA synthesis beginning in about 6 h. Labeled metaphases increased rapidly after 22 h without the appearance of an early rise in unlabeled metaphases. This suggests that the inhibitory effect of dibutyryl cyclic AMP is on the G1 phase of the cell cycle.