Basal-Cell Subpopulations and Cell-Cycle Kinetics In Human Epidermal Explant Cultures

Cultured human epidermal cells were studied by cell sorting and autoradiography after different ³H-thymidine (³H-dThd)-labelling procedures and after labelling with DNA precursors that are incorporated via salvage or de novo pathways. It was shown that ³H-dThd incorporation was the best measure of the rate of DNA replication. Dose-response experiments with pulse and continuous labelling revealed that all S- and G₂-phase cells were cycling, whereas some 20% of the cells stayed in G₁-phase for long periods of time. Most, if not all of these cells were probably non-proliferating differentiated keratinocytes. At least two subpopulations of S-phase cells could be discriminated on the basis of the rate of incorporation of DNA precursors. the difference in precursor incorporation did not seem to be caused by differences in nucleotide metabolism but rather to reflect true differences in the rate of DNA replication. Continuous labelling experiments showed that these subpopulations also were apparent in the G₁- and G₂-phases. Studies of the grain-count distribution revealed that cells that appeared to move rapidly through the S-phase moved slowly through the G₂-phase, and vice versa. Cells stained with acridine orange were subjected to a two-parameter analysis in the cell sorter by simultaneous measurement of the DNA and RNA fluorescence. Autoradiography of sorted cells revealed that, on average, cells with low RNA contents incorporated ³H-dThd at a higher rate than cells with high RNA contents.     

Factors affecting the formation of phytol and its incorporation into chlorophyll by homogenates of the leaves of the french bean Phaseolus vulgaris

The biosynthesis and phosphorylation of histone fractions were measured in synchronized CHO Chinese hamster cells arrested in late G₁ by hydroxyurea treatment. Hydroxyurea was found to inhibit the initiation of both DNA and histone synthesis, thus confirming the conclusion that it arrests cells in G₁ slightly before the $\text{G}{}_1\text{S}$ boundary. However, hydroxyurea did not inhibit the phosphorylation of histone f1 or histone f2a2. The phosphorylation of histone f1, which normally is absent in early G₁, begins 2 hr prior to DNA synthesis. In the presence of hydroxyurea, f1 phosphorylation occurs on schedule at this same time in G₁, resulting in significant G₁-phase f1 phosphorylation. This offers strong evidence that (a) f1 phosphorylation is not restricted to S phase; (b) “old” f1 which was synthesized in previous cell cycles is phosphorylated in G₁ before “new” f1 which is synthesized in S phase; and (c) G₁-phase f1 phosphorylation does not require new histone or new DNA synthesis.Histone f1 phosphorylation was observed to occur at accelerated rates in S phase over phosphorylation rates observed in late G₁-arrest. Data support the proposal that three different levels of f1 phosphorylation occur during the cell cycle: (1) a G₁-related phosphorylation of “old” f1; (2) an S-related phosphorylation of both “old” and “new” f1; and (3) a superphosphorylation of f1 associated with chromosome condensation during the G₂ to M transition. It is also possible that a limited proportion of f1 may be phosphorylated in G₁, perhaps at the initial DNA synthesis sites, and that an increased proportion of f1 is phosphorylated in S as DNA is synthesized. Similarities between the kinetics of histone f1 phosphorylation and the association of DNA with lipoprotein in synchronized control and hydroxyurea-treated cells suggest an involvement of f1 phosphorylation in cell-cycle-dependent chromatin structural changes.     

Hyaluronan substratum holds mesenchymal stem cells in slow-cycling mode by prolonging G<Subscript>1</Subscript> phase

We examined, in vitro, whether hyaluronan induces slow cycling in placenta-derived mesenchymal stem cells (PDMSCs) by comparing cell growth on a hyaluronan-coated surface with cell growth on a tissue-culture polystyrene surface. The hyaluronan-coated surface significantly downregulated the proliferation of PDMSCs, more of which were maintained in the G₀/G₁ phases than were cells on the tissue-culture polystyrene surface. Both PKH-26 labeling and BrdU incorporation assays showed that most PDMSCs grown on a hyaluronan-coated surface duplicated during cultivation indicating that the hyaluronan-coated surface did not inhibit PDMSCs from entering the cell cycle. Mitotic synchronization showed that the G₁-phase transit was prolonged in PDMSCs growing on a hyaluronan-coated surface. Increases in p27^Kip1 and p130 were the crucial factors that allowed hyaluronan to lengthen the G₁ phase. Thus, hyaluronan might be a promising candidate for maintaining stem cells in slow-cycling mode by prolonging their G₁-phase transit. This work was supported by research grant NSC95-2745-B-006-003-MY2 from the National Science Council, Taiwan, and by Landmark Project Grant A25, funded by the Taiwan Ministry of Education, from National Cheng Kung University.     

After X-irradiation a transient arrest of L929 cells inG 2-phase coincides with a rapid elevation of the level of O6-alkylguanine-DNA alkyltransferase

Summary Following X-irradiation of exponentially growing L929 cells two major phenomena have been observed. First, there was a delay in cell division which can be ascribed to the arrest of cells in theG ₂-phase (G ₂-block), and, second, the cellular content of the O⁶-alkylguanine-DNA alkyltransferase (AGT) was markedly increased. Flow cytometrical DNA-measurements revealed that cells began to accumulate in theG ₂-phase 4 h after irradiation (p.r.) irrespective of the X-ray dose, while both the fraction of cells blocked inG ₂ and the time period the cells persisted inG ₂ increased with the radiation dose. About 24 h past release from theG ₂-block the distribution of cells in the cell cycle was similar to that of untreated control cells. In comparison with control cells the AGT content in irradiated cells (4 Gy) was highest at about 48 h p.r. (3.4-fold increase). The highest ratio of increase in AGT was, however, observed to occur between about 4 and 13 h p.r. (2.6-fold increase). As shown by flow cytometrical measurements using a BrdUrd/DNA double labeling technique, this rapid primary increase in AGT coincides very well with the entrance of cells into theG ₂-phase. This indicates that the cellular AGT content in X-irradiated (parental) cells started to exceed the basal level at the beginning of theG ₂-phase, but not before or during theS-phase. Once the AGT level was elevated it continued to increase for 2 to 3 cell doubling times.Paper given at the Workshop Molecular Radiation Biology. German Section of the DNA Repair Network, München-Neuherberg, 21.-23.3.1990     

Fission Yeast Ste9, a Homolog of Hct1/Cdh1 and Fizzy-related, Is a Novel Negative Regulator of Cell Cycle Progression during G1-Phase

When proliferating fission yeast cells are exposed to nitrogen starvation, they initiate conjugation and differentiate into ascospores. Cell cycle arrest in the G₁-phase is one of the prerequisites for cell differentiation, because conjugation occurs only in the pre-Start G₁-phase. The role of ste9⁺ in the cell cycle progression was investigated. Ste9 is a WD-repeat protein that is highly homologous to Hct1/Cdh1 and Fizzy-related. The ste9 mutants were sterile because they were defective in cell cycle arrest in the G₁-phase upon starvation. Sterility was partially suppressed by the mutation in cig2 that encoded the major G₁/S cyclin. Although cells lacking Ste9 function grow normally, the ste9 mutation was synthetically lethal with the wee1 mutation. In the double mutants of ste9 cdc10^ts, cells arrested in G₁-phase at the restrictive temperature, but the level of mitotic cyclin (Cdc13) did not decrease. In these cells, abortive mitosis occurred from the pre-Start G₁-phase. Overexpression of Ste9 decreased the Cdc13 protein level and the H1-histone kinase activity. In these cells, mitosis was inhibited and an extra round of DNA replication occurred. Ste9 regulates G₁ progression possibly by controlling the amount of the mitotic cyclin in the G₁-phase.     

DIFFERENTIATION OF NEUROBLASTOMA CELLS IN CULTURE

1. An elevation of the intracellular level of cyclic AMP in neuroblastoma cells by prostaglandin E₁ by an inhibitor of cyclic AMP phosphodiesterase, or by analogues of cyclic AMP irreversibly induces many differentiated functions which are characteristic of mature neurones. These include formation of long neurites, increase in size of soma and nucleus associated with a rise in total RNA and protein contents, increase in activities of specific neural enzymes, loss of malignancy, increase in sensitivity of adenylate cyclase to catecholamines and blockade of cells in G₁-stage of the cell cycle. 2. Other agents, including serum-free medium, X-irradiation, 6-thioguanine, cytosine arabinoside, methotrexate, 5-bromodeoxyuridine, nerve growth factor, glial extract and hypertonic medium can induce some of the differentiated functions which are induced by high intracellular cyclic AMP. 3. Morphological differentiation and differentiated biochemical functions can each be expressed in the absence of the other. 4. Many of the responses of normal embryonic nerve cells to cyclic AMP are similar to those of neuroblastoma cells. 5. A working hypothesis for the malignancy of nerve cells has been proposed. This states that an abnormal regulation of cyclic AMP phosphodiesterase activity which allows the expression of high amounts of this enzyme in neuroblastoma cells, may be one of the early lesions during a malignant transformation of nerve cells. 6. A new experimental therapeutic model for the treatment of neuroblastoma is proposed. This involves the administration of sodium butyrate followed by the injection of l -dihydroxyphenylalanine (l-dopa) and prostaglandin E₁ in the presence of cyclic AMP phosphodiesterase inhibitor. 7. Recent studies have elucidated the control mechanisms of some differentiated functions in neuroblastoma cells. Cyclic AMP may become an important biological tool to probe the regulation and expression of many other differentiated functions in these cells. In addition to neuroblastoma cells, other neuronal culture systems are now available for investigating the problems of differentiation and maturation in nerve cells.     

REGULATION OF INITIATION OF DNA SYNTHESIS IN CHINESE HAMSTER CELLS : II. Induction of DNA Synthesis and Cell Division by Isoleucine and Glutamine in G1-Arrested Cells in Suspension Culture

Suspension cultures of Chinese hamster cells (line CHO), which had stopped dividing and were arrested in G₁ following growth to high cell concentrations in F-10 medium, could be induced to reinitiate DNA synthesis and to divide in synchrony upon addition of the appropriate amounts of isoleucine and glutamine. Both amino acids were required to initiate resumption of cell-cycle traverse. Deficiencies in other amino acids contained in F-10 medium did not result in accumulation of cells in G₁, indicating a specific action produced by limiting quantities of isoleucine and glutamine. In the presence of sufficient glutamine, approximately 2 x 10^-6 M isoleucine was required for all cells to initiate DNA synthesis in a population initially containing 1.5 x 10⁵ cells/ml. Under similar conditions, about 4 x 10^-6 M isoleucine was required for all G₁-arrested cells to progress through cell division. In contrast, 1 x 10^-4 M glutamine was necessary for maximum initiation of DNA synthesis in G₁ cells, along with sufficient isoleucine. A technique for rapid production of G₁-arrested cells is described in which cells from an exponentially growing population placed in F-10 medium deficient in both isoleucine and glutamine or isoleucine alone accumulated in G₁ after 30 hr.     

The induction of chromosome-type aberrations in G1 by methyl methanesulfonate and 4-nitroquinoline-N-oxide,and the non-requirement of an S-phase for their production

Human lymphocyte were treated in G₁ with 4-nitroquinoline-N-oxide (4NQO) and methyl methanesulfonate (MMS) and then incubated in the presence or absence of cytosine arabinoside (ara-C). There was an increase in aberration frequency in those cells incubated with ara-C compared with those treated with 4NQO or MMS alone. This increase was restricted to chromosome-type aberrations. When cells were treated in G₂ with 4NQO and then incubated with ara-C until fixation, there was an increase in deletions compared with cells treated with 4NQO alone. No exchange aberrations were observed following any treatment even when deletion frequencies were high, as in the case with 4NQO plus ara-C treatment. These results suggest that ara-C can inhibit the repair of DNA damage induced by 4NQO and MMS that is converted into aberrations. They also show that the terms “S-dependent” and “S-independent” used to describe the modes of action of chemical clastogens are not valid.     

Reversible (G0) and nonreadily reversible (Q) noncycling cells in human peripheral blood

PHA-stimulated human lymphoctes (normal-resting-proliferating) at 0, 24, 48, 72, and 144 h were studied with Acridine Orange (AO) staining. By viable cell sorting, by subsequent subculturing, and by use of biochemical, biophysical, and immunological assays, not only have the G₀ resting and G₁ (cycling) cell cycle phases been objectively characterized, but a separate subpopulation of quiescent cells that are functionally viable and deeply committed to nonproliferation, the Q cells, has been identified. Multiparameter cytofluorimetric analysis, methyl¹⁴C-thymidine incorporation, automated image analysis, and mitogen stimulation studies have shown that the “Q” cell, compared to the “G₀” resting but easily recruitable cell, exhibits quite lower red and green AO emission, possesses 2c to 4c DNA content (rather than only 2c), has a higher average optical density, and is either nonrecruitable or recruitable-with-difficulty in PHA-stimulated lymphocyte cultures.     

Differential chromosomal radiosensitivity within the first G1-phase of the cell cycle of early-dividing human leukocytes in vitro after stimulation with PHA

Summary Human leukocyte cultures were irradiated with 200 R X-rays before the addition of phytohemagglutinin (PHA) in the G₀-stage and at different times up to 25 h within the first G₁-phase of the cell cyle after the addition of PHA. The results of the analysis of chromosomal aberrations show that the frequencies of dicentric chromosomes increase significantly when leukocytes leave the G₀-stage, reaching a maximum yield of aberrations about halfway through the first G₁-phase. After that, toward the end of the G₁-phase, the frequencies of dicentric chromosomes decrease again, to a level similar to that found in the G₀-stage. Different possible explanations for the differential chromosomal radiosensitivity of human leukocytes within the first poststimulation G₁-phase are discussed.     

Tubulin Polymerization Promoting Protein 1 (Tppp1) Phosphorylation by Rho-associated Coiled-coil Kinase (Rock) and Cyclin-dependent Kinase 1 (Cdk1) Inhibits Microtubule Dynamics to Increase Cell Proliferation

Tubulin polymerization promoting protein 1 (Tppp1) regulates microtubule (MT) dynamics via promoting MT polymerization and inhibiting histone deacetylase 6 (Hdac6) activity to increase MT acetylation. Our results reveal that as a consequence, Tppp1 inhibits cell proliferation by delaying the G₁/S-phase and the mitosis to G₁-phase transitions. We show that phosphorylation of Tppp1 by Rho-associated coiled-coil kinase (Rock) prevents its Hdac6 inhibitory activity to enable cells to enter S-phase. Whereas, our analysis of the role of Tppp1 during mitosis revealed that inhibition of its MT polymerizing and Hdac6 regulatory activities were necessary for cells to re-enter the G₁-phase. During this investigation, we also discovered that Tppp1 is a novel Cyclin B/Cdk1 (cyclin-dependent kinase) substrate and that Cdk phosphorylation of Tppp1 inhibits its MT polymerizing activity. Overall, our results show that dual Rock and Cdk phosphorylation of Tppp1 inhibits its regulation of the cell cycle to increase cell proliferation.     

ON THE EXISTENCE OF A Go-PHASE IN THE CELL CYCLE

The model is based on the assumption that the cell cycle contains a G_o-phase which cells leave randomly with a constant probability per unit time, γ. After leaving the G_o-phase, the cells enter the C-phase which ends with cell division. The C-phase and its constituent phases, the‘true’G₁-phase, the S-phase, the G₂-phase and mitosis are assumed to have constant durations of T, T₁T_s, T₂ and T_m, respectively. For renewal tissue it is assumed that the probability per unit time of being lost from the population is a constant for all cells irrespective of their position in the cycle. The labelled mitosis curve and labelling index for continuous labelling are derived in terms of γ, T, and T_s. The model generates labelled mitosis curves which damp quickly and reach a constant value of twice the initial labelling index, if the mean duration of the G_o-phase is sufficiently long. It is shown that the predicted labelled mitosis and continuous labelling curves agree reasonably well with the experimental curves for the hamster cheek pouch if T has a value of about 60 hr. Data are presented for the rat dorsal epidermis which support the assumption that there is a constant probability per unit time of a cell being released from the Go-phase.     

Centriole duplication in PE (SPEV) cells starts before the beginning of the DNA replication

Centrosome includes two centrioles and is a structural basis of mitotic spindle pole. Duplication of this organelle and doubling of chromosomes quantity during DNA replication are two principal events of cell cycle in the course of preparation for cell division. In this work, cells of pig kidney embryonic cell line PE (SPEV) were individually monitored after mitosis and procentriole appearance was detected by electron microscopy as soon as 5–6 h after mitosis. This period was 1–2 h shorter than minimal duration of G₁-phase in PE cell line. Ultrastructural analysis of centrosomes in the cells with known “cell cycle age” in combination with autoradiography study of the same cells using ³H-thimidine directly confirmed that duplication of centrioles started earlier than cells entered in S-phase of cell cycle, i.e., preceded the DNA replication.     

Role of voltage‐gated potassium channels in the fate determination of embryonic stem cells

Embryonic stem cells (ESCs) possess two unique characteristics: self‐renewal and pluripotency. In this study, roles of voltage‐gated potassium channels (K_v) in maintaining mouse (m) ESC characteristics were investigated. Tetraethylammonium (TEA⁺), a K_v blocker, attenuated cell proliferation in a concentration‐dependent manner. Possible reasons for this attenuation, including cytotoxicity, cell cycle arrest and differentiation, were examined. Blocking K_v did not change the viability of mESCs. Interestingly, K_v inhibition increased the proportion of cells in G₀/G₁ phase and decreased that in S phase. This change in cell cycle distribution can be attributed to cell cycle arrest or differentiation. Loss of pluripotency as determined at both molecular and functional levels was detected in mESCs with K_v blockade, indicating that K_v inhibition in undifferentiated mESCs directs cells to differentiate instead of to self‐renew and progress through the cell cycle. Membrane potential measurement revealed that K_v blockade led to depolarization, consistent with the role of K_v as the key determinant of membrane potential. The present results suggest that membrane potential changes may act as a “switch” for ESCs to decide whether to proliferate or to differentiate: hyperpolarization at G₁ phase would favor ESCs to enter S phase while depolarization would favor ESCs to differentiate. Consistent with this notion, S‐phase‐synchronized mESCs were found to be more hyperpolarized than G₀/G₁‐phase‐synchronized mESCs. Moreover, when mESCs differentiated, the differentiation derivatives depolarized at the initial stage of differentiation. This investigation is the first study to provide evidence that K_v and membrane potential affect the fate determination of ESCs. J. Cell. Physiol. 224:165–177, 2010 © 2010 Wiley‐Liss, Inc.     

The metabolism of histone fractions. VI. Differences in the phosphorylation of histone fractions during the cell cycle

Phosphorylation of histone fractions in the presence and absence of DNA synthesis was measured using the new “isoleucine-limiting” method for synchronizing Chinese hamster cells in early G₁-phase. Using preparative electrophoresis, histone f1 phosphorylation was found to be dependent upon cell-cycle position, being absent in G₁-arrested and G₁-traversing cells and active in the S-phase. The absence of f1 phosphorylation in G₁-arrested cells, which are known to exhibit f1 turnover, indicates that f1 phosphorylation is not an obligatory part of the f1 turnover process. In contrast to histone f1, it was found that histone f2a2 phosphorylation is independent of cell-cycle position, occurring with equal magnitude in the G₁-traversing state when DNA synthesis is essentially absent and in the S-phase when DNA synthesis is active. When cells were arrested in the G₁-state by isoleucine deprivation, f2a2 phosphorylation continued to be active, occurring at 56% of the rate observed in the G₁-traversing state. These results indicate that phosphorylation of histone f2a2 is independent of f2a2 synthesis, independent of DNA synthesis, and independent of histone f1 phosphorylation. Because f2a2 is actively phosphorylated in G₁-arrested cells known to be active in the synthesis of various types of RNA (including messenger) as well as in G₁-traversing and S-phase cells, we feel that phosphorylation of histone f2a2 should continue to be considered in models concerning activation of DNA template activity.     

Cytophotometric analysis of glycogen,protein and DNA of a glycogen-storing rat hepatoma (N13) cell line

This study examines the behavior of glycogenstoring rat hepatoma (N13) in vitro using cytophotometric techniques. A significant increase in glycogen is observed in these cells after 30 min incubation in a buffered solution containing 0.1 mM glucose, that is 80 times lower than the physiological glucose concentration in rat blood. N13 hepatoma cells grow exponentially in culture using RPMI 1640 tissue culture medium supplemented with 10% fetal bovine serum. During the first day in culture these cells store a large amount of glycogen and this increase is also observed in serum-free cultures. In more prolonged cultures the amount of glycogen per cell gradually becomes lower, although the culturing conditions are maintained. Similar variations of protein are also observed during the initial period of culture. DNA distribution does not show significant changes, although in serum-free cultures an increase in the proportion of cells in S and G₂/M phases is observed. The addition of glucagon, epinephrine and cyclic AMP derivatives to serum-free cultures does not impede the storage of glycogen. Nevertheless, addition of either 2 mM N⁶,O²-dibutyryl cyclic AMP or 0.1 mM 8-(4-chlorophenylthio)-cyclic AMP blocks the cell cycle at G₀/G₁ and glycogen content does not decrease after the first day in culture. We believe that this cell line offers an appropriated model to study glycogen metabolism and its involvement in the neoplastic process.     

PRIMORDIAL GERM CELL-DERIVED MOUSE EMBRYONIC GERM (EG) CELLSIN VITRORESEMBLE UNDIFFERENTIATED STEM CELLS WITH RESPECT TO DIFFERENTIATION CAPACITY AND CELL CYCLE DISTRIBUTION

Embryonic germ (EG) cells of line EG-1 derived from mouse primordial germ cells were investigated for theirin vitrodifferentiation capacity. By cultivation as embryo-like aggregates EG-1 cells differentiated into cardiac, skeletal muscle and neuronal cells accompanied by the expression of tissue-specific genes and proteins as shown by RT-PCR analysis and indirect immunofluorescence. In comparison to embryonic stem (ES) cells of line D3 the efficiency of differentiation into cardiac and muscle cells was comparatively low, whereas spontaneous neuronal differentiation was more efficient than in D3 cells. Furthermore, the distribution of cell cycle phases as a parameter for the differentiation state was analysed in undifferentiated EG cells and ES cells and compared to data obtained for embryonic carcinoma (EC) cells of line P19 and differentiated, epithelioid EPI-7 cells. Flow cytometric analysis revealed similar cell cycle phase distributions in EG, EC and ES cells. In contrast, the somatic differentiated EPI-7 cells showed a longer G₁-phase and shorter S- and G₂/M-phases. Together, our results demonstrate that the differentiation state and capacity of EG cellsin vitroresemble that of totipotent ES cells.     

The positive control of cell proliferation by the interplay of calcium ions and cyclic nucleotides. A review

Conclusion Calcium, cyclic AMP, and cyclic GMP do not seem to be involved in proliferative activation of postmitotic differentiated cells. Instead, they are intracycle regulators, and we propose the following working model of their control of the initiation of DNA synthesis. While a role for cyclic GMP cannot yet be defined, a brief postmitotic burst of its synthesis might serve to prevent certain activated cells (e.g. 3T3 mouse cells) from being diverted into a nonproliferating (but still activated) G₀ state (Figs. 1 and 17). In a latter part of the G₁ phase, something happens to stimulate briefly the synthesis of cyclic AMP which, in turn, drives calcium ions from the mitochondria into the cytosol to activate newly synthesized thymidylate synthetase (or other primed enzymic assemblies) (Fig. 1). Having “turned on” their target enzymes, the accumulated cyclic AMP is destroyed and the excess calcium ions are reaccumulated by the mitochondria to avoid interfering with succeeding reactions. This model predicts that persistent changes in cyclic AMP metabolism and the respiration-linked, calcium-accumulating (ion-buffering) activity of mitochondria may be responsible for the sustained growth of tumors. Issued as NRCC No. 14974.