The comparative cell cycle and metabolic effects of chemical treatments on root tip meristems. III. Chlorsulfuron

Intact and excised cultured pea roots (Pisum sativum L. cv Alaska) were treated with chlorsulfuron at concentrations ranging from 2.8 ×10^?4 M to 2.8×10^?6 M. At all concentrations this chemical was demonstrated to inhibit the progression of cells from G₂ to mitosis (M) and secondarily from G₁ to DNA synthesis (S). The S and M phases were not directly affected, but the transition steps into those phases were inhibited. Total protein synthesis was unaffected by treatment of intact roots with 2.8×10^?6 M chlorsulfuron. RNA synthesis was inhibited by 43% over a 24-h treatment period. It is hypothesized that chlorsulfuron inhibits cell cycle progression by blocking the G₂ and G₁ transition points through inhibition of cell cycle specific RNA synthesis.     

Metabolism of benzene and phenol in macrophages in vitro and the inhibition of RNA synthesis by benzene metabolites

Benzene may affect hemopoiesis by damaging the bone marrow stroma that provides the microenvironment for hemopoiesis. A possible target of benzene toxicity in the stroma is the macrophage, which is a major source of protein factors required for the proliferation and differentiation of progenitor cells. As an initial approach towards understanding whether benzene inhibits hemopoietic factor production in bone marrow stroma, the metabolism of benzene and phenol has been studied and the effect of benzene and its metabolites on macrophage RNA synthesis has been examined. Benzene is not metabolized in macrophages but phenol, the major metabolite of benzene in bone marrow, is converted by peroxidase in the macrophage to both free metabolites and species which covalently bind to cellular macromolecules. Benzene and its metabolites inhibited RNA synthesis in a dose-dependent manner, with 50% inhibitory concentrations of 5 × 10^–3M for benzene, 2.5 × 10^–3 M for phenol, 2.5 × 10^–5 M for hydroquinone, and 6 × 10^–6 M for p-benzoquinone; this inhibition was not attributable to loss of cell viability. Benzene, possibly by an inhibition of uridine transport into macrophages, and phenol, by its conversion to covalently binding species, inhibit RNA synthesis in macrophages and thus may inhibit the synthesis of colony stimulating factors required for hemopoiesis.Abbreviations CFU-G / M colony forming unit-granulocyte / macrophage - FCS fetal calf serum - IC₅₀ molar concentration causing 50% inhibition - PBS phosphate buffered saline     

Amino Acid Metabolism of Lemna minor L. : II. Responses to Chlorsulfuron

Chlorsulfuron, an inhibitor of acetolactate synthase (EC 4.1.3.18) (TB Ray 1984 Plant Physiol 75: 827-831), markedly inhibited the growth of Lemna minor at concentrations of 10⁻⁸ molar and above, but had no inhibitory effects on growth at 10⁻⁹ molar. At growth inhibitory concentrations, chlorsulfuron caused a pronounced increase in total free amino acid levels within 24 hours. Valine, leucine, and isoleucine, however, became smaller percentages of the total free amino acid pool as the concentration of chlorsulfuron was increased. At concentrations of chlorsulfuron of 10⁻⁸ molar and above, a new amino acid was accumulated in the free pool. This amino acid was identified as α-amino-n-butyrate by chemical ionization and electron impact gas chromatography-mass spectrometry. The amount of α-amino-n-butyrate increased from undetectable levels in untreated plants, to as high as 840 nanomoles per gram fresh weight (2.44% of the total free pool) in plants treated with 10⁻⁴ molar chlorsulfuron for 24 hours. The accumulation of this amino acid was completely inhibited by methionine sulfoximine. Chlorsulfuron did not inhibit the methionine sulfoximine induced accumulations of valine, leucine, and isoleucine, supporting the idea that the accumulation of the branched-chain amino acids in methionine sulfoximine treated plants is the result of protein turnover rather than enhanced synthesis. Protein turnover may be primarily responsible for the failure to achieve complete depletion of valine, leucine, and isoleucine even at concentrations of chlorsulfuron some 10⁴ times greater than that required to inhibit growth. Tracer studies with ¹⁵N demonstrate that chlorsulfuron inhibits the incorporation of ¹⁵N into valine, leucine, and isoleucine. The α-amino-n-butyrate accumulated in the presence of chlorsulfuron and [¹⁵N]H₄⁺ was heavily labeled with ¹⁵N at early time points and appeared to be derived by transamination from a rapidly labeled amino acid such as glutamate or alanine. We propose that chlorsulfuron inhibition of acetolactate synthase may lead to accumulation of 2-oxobutyrate in the isoleucine branch of the pathway, and transamination of 2-oxobutyrate to α-amino-n-butyrate by a constitutive transaminase utilizing either glutamate or alanine as α-amino-N donors.     

MICRONUCLEAR RNA SYNTHESIS IN PARAMECIUM CAUDATUM

In a generation time of 8 hr in Paramecium caudatum, the bulk of DNA synthesis detected by thymidine-³H incorporation takes place in the latter part of the cell cycle. The micronuclear cycle includes a G₁ of 3 hr followed by an S period of 3–3½ hr. G₂ and division occupies the remaining period of the cycle. Macronuclear RNA synthesis detected by 5'-uridine-³H incorporation is continuous throughout the cell cycle. Micronuclear RNA synthesis is restricted to the S period. Ribonuclease removes 80–90% of the incorporated label. Pulse-chase experiments showed that part of the RNA is conserved and released to the cytoplasm during the succeeding G₁ period.     

Some effects of 2,4,5-trichlorophenoxyacetic acid on the mitotic cycle of lateral root apical meristems of Vicia faba

Roots of Vicia faba were given a one hour pulse label with. ³H-TdR (1 C/ml), either before or after a three hour treatment with a 10^–5 M solution of 2,4,5-trichlorophenoxyacetic acid (TCPA). The durations of the various phases of the mitotic cycle were derived from labeled prophase curves, prepared from autoradiographs of lateral root apical meristems. — TCPA was found to lengthen the duration of the mitotic cycle, primarily because it extended the duration of the period of DNA synthesis (S), though post-synthetic interphase (G₂) was also longer. No measurements could be made with respect to the duration of presynthetic interphase (G₁), because of rapid changes in the lengths of the G₂ and S periods following treatment. — As well as extending the duration of S, TCPA treatment also resulted in at least an initial increase in the rate of DNA synthesis and a decrease in the actual number of cells in S. These results have been discussed with respect to the control of the organization of the root apical meristem.Supported by a grant from the Assistant Professor Research Fund of the University of Missouri.     

Effects and Absorption of Sethoxydim in Cell Cycle Progression of Corn (Zea mays) and Pea (Pisum sativum)

The mechanisms of selective herbicidal action of sethoxydim were investigated by using cultured root tips of corn (Zea mays L. cv Goldencrossbantam) and pea (Pisum sativum L. cv Alaska). Meristematic cells in the cultured roots were arrested in G₁ and G₂ of the cell division cycle by sucrose starvation and resumed growth and cell division (proliferation) when sucrose was provided. Corn root growth after sucrose addition was inhibited by sethoxydim at concentrations of 0.01 micromolar and greater when roots were treated in the presence of sucrose but was not inhibited at 10 micromolar sethoxydim when they were treated during sucrose starvation. Greater absorption of [¹⁴C]sethoxydim into the meristematic region of corn roots was observed when cells were in proliferative condition but not when they were arrested by sucrose starvation, whereas no greater absorption of the herbicide into pea meristems was observed in either growth condition. In the cell cycle study, greater absorption of [¹⁴C]sethoxydim into the corn root meristem was observed at a certain limited time before S (DNA synthesis) stage. The physiological effects and the greater absorption of sethoxydim clearly depended on cell cycle progression of corn root meristem, whereas fatty acid synthesis, as well as its inhibition by sethoxydim, was not associated with either cell cycle progression or greater absorption of the herbicide.     

Experession and integration of genes introduced into highly synchronized plant protoplasts

Summary Chimaeric genes containing the chloramphenicol acetyltransferase (CAT) coding sequence were introduced into protoplasts of suspension-cultured tobacco cells using improved conditions of electroporation (Okada et al. 1986). CAT activity became detectable in the protoplasts within 3 h, was maximal during a period of 18–36 h after electroporation, and then declined gradually. Alpha-amanitin added to the medium abolished the transient expression of the CAT gene. The closed circular form of input DNA was as effective as the linear form for the transient expression. The suspension culture was treated with aphidicolin, and S, G₂, M and G₁ phases were identified in the highly synchronized cell cycle obtained by releasing the cells from the inhibition of DNA synthesis. When a chimacric CAT gene was introduced into M phase protoplasts prepared from the synchronized culture, the transient expression of the CAT gene was 3–4 times higher than when it was introduced into protoplasts of other cell cycle phases. The frequency of stable transformation with a chimaeric neomycin phosphotransferase II gene was studied using the same system. G-418-resistant transformants were obtained from M phase protoplasts at frequencies 2–8 times those obtained from protoplasts at other cell cycle phases. The results indicate that the absence of the nuclear membrane in mitotic cells favours delivery to the nucleus of exogenous DNA introduced into the cytoplasm.     

Ethylene metabolism in Pisum sativum L.: Kinetic parameters,the effects of propylene,silver and carbon dioxide and comparison with other systems

The kinetic parameters of in vivo ethylene metabolism by seedlings of Pisum sativum L. cv. Alaska have been determined. The oxidation of ethylene to CO₂, (Ox) and the incorporation of ethylene into the tissue (TI) were both shown to display Michaelis-Menten kinetics (K_m Ox = 0.9 × 10^–6 M liquid phase, V_max Ox = 2.4 × 10^–10 moles g^– dry mass h^–1 K_m TI = 1.6 × 10^–6 M liquid phase, V_max TI = 4.5 × 10^–10 moles g^–1 dry mass h^–1). Propylene competitively inhibited both Ox (K_i = 7.0 × 10^–6 M) and TI (K_i = 3.7 × 10^–7 M). A system comparable to Ox was absent from imbibed cotyledons of Vicia faba L. cv. Aquadulce even at saturating concentrations of ethylene similar to those used in kinetic analysis on Pisum. Silver ions were shown to inhibit TI but promoted Ox, while carbon dioxide inhibited Ox but promoted TI. Kinetic data on both these effects are presented. Data on the effect of a range of concentrations of CO₂ on TI and Ox are also presented.To whom editorial correspondence should be sent     

Changes in the content of condensed chromatin during the cell cycle in antheridial filaments ofChara vulgaris L. as related to DNA and RNA synthesis,3H actinomycin D binding and RNA polymerase activity

Summary Using light microscopic autoradiography it was found that in the middle S and G₂ phases of the cell cycle in antheridial filaments (S + G₂ + M type) ofChara vulgaris L., an intensified RNA synthesis took place. The process was correlated with enhanced binding of³H actinomycin D to the DNA template, increased RNA polymerase activity, and enhanced chromatin decondensation.This work was supported by the Polish Academy of Sciences within the project 09.7.3.1.4.     

Intranucleolar visualization of nucleic acids and acidic proteins in inhibited and reactivated pea cotyledonary buds

Summary Nuclease-colloidal gold complexes and silver staining were used to visualize intranucleolar nucleic acids and argyrophilic proteins of the nucleolar organizers in bud cotyledonary cells ofPisum sativum. In the G_0–1 inhibited bud, a few RNA molecules were detected in the fibrillar component and in the unique fibrillar centre, close to the boundary with the fibrillar component of the nucleolus. DNA was present in the fibrillar component, in the fibrillar centre and in a few fibres crossing the perinucleolar halo. The acidic proteins were localized at the periphery of the fibrillar component but they were also present in the unique fibrillar centre. In the reactivated bud, RNA was particularly concentrated in the granular component and along fibres crossing the perinucleolar halo; a few RNA molecules were also detected at the boundary between the small fibrillar centres and the fibrillar component. DNA was localized in the same nucleolar component as in the inhibited bud, but it was distributed between several fibrillar centres. Acidic proteins coated these DNA loci. In the inhibited and reactivated bud connections between nucleolar DNA containing structures were displayed. The data are discussed in relation to the present knowledge of the functional architecture of the nucleolus.Abbreviations DNA deoxyribonucleic acid - DNase deoxyribonuclease - G_0–1 phase G₁ phase of the cell cycle indefinitely prolonged - PEG polyethylene glycol - RNA ribonucleic acid - RNase ribonuclease - S and G₂ phases synthetic and postsynthetic phases of the cell cycle - SPB saline phosphate buffer     

RNA synthesis in synchronously growing populations of HeLa S3 cells. I. Rate of total RNA synthesis and its relationship to DNA synthesis

The rate of RNA synthesis in synchronously growing HeLa S3 cells was determined as a function of position in the cell generation cycle. Measurements throughout the cycle of both the rate of incorporation of radioactively-labeled uridine and of the total amount of RNA indicate that (1) the rate of RNA synthesis is constant (or increases only slightly) during G₁, approximately doubles during the first half of S, and then remains constant during the remainder of S and G₂, and (2) cells attain the average G₁ rate of RNA synthesis very early in G¹, and maintain the average G₂ rate until mitosis. If the initiation of DNA synthesis is blocked, the acceleration of RNA synthesis is markedly reduced or eliminated. Further experiments in which DNA synthesis was inhibited at different times in S, or to varying degrees from the beginning of S, suggest that the extent to which RNA synthesis is accelerated depends on the amount of DNA duplicated. These data also indicate that duplication of the first half, and in particular the first few per cent, of the DNA complement results in a disproportionate acceleration of RNA synthesis. The possibility that fluctuations in the sizes of precursor pools may lead to misinterpretation of labeled-uridine incorporation data was examined. Experiments indicate that in this system pool fluctuations do not cause invalid measures of RNA synthesis. It is concluded that RNA synthesis occurs throughout interphase, but undergoes a two-fold increase in rate which is dependent on the duplication of DNA.     

2,6-Diisopropylphenol,a general anesthetic,inhibits glutamate action on rat synaptosomes

2,6-diisopropylphenol (propofol), a general intravenous anesthetic, inhibits the glutamate-dependent Ca²⁺ entry in rat synaptosomes with an approximate IC₅₀ of 3.0×10^–5 M.Propofol, at concentrations above 10^–6M, also inhibits the ATP-dependent uptake of glutamate in the presence of Ca²⁺, with an approximate IC₅₀ of 3.5×10^–5M, while it only has a slight inhibitory effect on the release of glutamate. The ouabain-insensitive synaptosomal ATPase is strongly inhibited by propofol, with an IC₅₀ of about 2.5×10^–6M, at concentrations which do not affect the luciferase system.     

Evidence for the Active and Passive Chemotropisms in Roots

Changes in the elongation of root cells during the negative (away from the salt) or positive (towards the salt) chemotropic bending of roots induced by the unilateral application of agar blocks (1 mm³) with 10^–3and 10^–2M Cd(NO₃)₂solutions to the meristem zone of the root were studied. The root bending was not accompanied by differential changes in the number of cells that elongated during the 3-h period of chemical stimulation. The bending was only due to differential changes in the cell elongation rates. In most chemically stimulated roots, both concentrations of Cd(NO₃)₂solutions inhibited cell elongation at the stimulated and nonstimulated sides. Cell elongation was inhibited by 10^–2M Cd(NO₃)₂mainly on the stimulated side of the roots, hence, the roots bent towards the salt. On the contrary, 10^–3M Cd(NO₃)₂inhibited cell elongation mainly at the nonstimulated side of the roots. As a result, the roots bent away from the salt, i.e., in the direction opposite to that expected in the case of the direct inhibition of cell growth by Cd(NO₃)₂. It is concluded that the root chemotropisms induced by the above two Cd(NO₃)₂concentrations are, correspondingly, of a passive or active nature.     

Modulation of G(2) arrest enhances cell death induced by the antitumor 1-nitroacridine derivative,Nitracrine

Nitracrine (Ledakrin) is an antitumor drug which is activated by cellular enzymes and binds covalently to DNA. Previous studies have shown that covalent binding and crosslinking of DNA is associated with the cytotoxic and antitumor activities of this compound. In this study, cell cycle perturbations, effects on DNA synthesis and the cell death process initiated by Nitracrine were studied in murine leukemia L1210 cells. We show that exposure of L1210 cells to Nitracrine at the IC₉₉ concentration delayed progression through the S phase and transiently arrested cells in G₂/M as found by flow cytometry. Higher drug concentration (2 × IC₉₉) inhibited cell cycle progression in the S phase and induced rapid cell death. Both studied concentrations of the drug produced different effects on DNA synthesis as determined by bromodeoxyuridine incorporation, with a delay in the S phase progression at EC₉₉ concentration and irreversible arrest in early S phase at the higher dose (2 × IC₉₉). At both concentrations of Nitracrine cell death occurred preferentially in the S phase as revealed by the TUNEL assay. When cells treated with the drug for 4 hours were post-incubated in the presence of 1 mM caffeine this led to rapid cell death and suppression of the G₂ arrest. This was associated with a about 10-fold increase in the cytotoxicity of Nitracrine. Similar effects were observed for another DNA crosslinking agent, cis-platinum, and to a lesser extent, for DNA topoisomerase I inhibitor, camptothecin. Together, our studies show that suppression of G₂ arrest induced by Nitracrine greatly enhances its cytotoxicity toward L1210 cells.     

Skp1-Cul1-F-box Ubiquitin Ligase (SCFβTrCP)-mediated Destruction of the Ubiquitin-specific Protease USP37 during G2-phase Promotes Mitotic Entry

Ubiquitin-mediated proteolysis is a key regulatory process in cell cycle progression. The Skp1-Cul1-F-box (SCF) and anaphase-promoting complex (APC) ubiquitin ligases target numerous components of the cell cycle machinery for destruction. Throughout the cell cycle, these ligases cooperate to maintain precise levels of key regulatory proteins, and indirectly, each other. Recently, we have identified the deubiquitinase USP37 as a regulator of the cell cycle. USP37 expression is cell cycle-regulated, being expressed in late G₁ and ubiquitinated by APC^Cdh1 in early G₁. Here we report that in addition to destruction at G₁, a major fraction of USP37 is degraded at the G₂/M transition, prior to APC substrates and similar to SCF^βTrCP substrates. Consistent with this hypothesis, USP37 interacts with components of the SCF in a βTrCP-dependent manner. Interaction with βTrCP and subsequent degradation is phosphorylation-dependent and is mediated by the Polo-like kinase (Plk1). USP37 is stabilized in G₂ by depletion of βTrCP as well as chemical or genetic manipulation of Plk1. Similarly, mutation of the phospho-sites abolishes βTrCP binding and renders USP37 resistant to Plk1 activity. Expression of this mutant hinders the G₂/M transition. Our data demonstrate that tight regulation of USP37 levels is required for proper cell cycle progression.     

Expression of the mitochondrial genome in HeLa cells. V. Transcription of mitochondrial DNA in relationship to the cell cycle

An investigation of the rate of incorporation of [5-³H]ur dine into mitochondrial RNA in synchronized HeLa cells in different phases of the cell cycle has revealed a considerable acceleration of this incorporation in cells in S and especially in G₂ phase. An analysis of the labeling of the intramitochondrial UTP pool has shown that this acceleration reflects a true increase in the rate of synthesis of mitochondrial RNA: this increase is considerably greater than can be accounted for by the expected doubling of mit-DNA^‡ templates during the S and G₂ phases.     

The differentiation of T-lymphocytes. III. The behaviour of subpopulations of mouse thymus cells in short-term cell culture.

Seven human cultured lymphoblastoid cell lines (CLL) were divided into two major groups based on studies of their cell cycle characteristics and surface Ig. CLL I (lines CL, MW, HH and TM) had generation times ranging from 25–40 hr, S phase times of 10–12 hr, G₂ + M times of 6–8 hr, and demonstrated sharp differences between the percentage of SIg(+) cells in different phases of the cell cycle. Line TM was particularly discordant with the highest percentage of SIg(+) cells in G₂ + M. CLL II (lines PS, JR and HT) demonstrated generation times ranging from 18–21 hr, S phase times of 7–10 hr and G₂ + M phase times of 2 hr. In this second group, two of the three CLLs had no differences between cells taken from different points of the cell cycle. DNA synthesis and cell density could not be correlated with either of the above major parameters, i.e. cell cycle times or SIg expression. The results suggest that human CLLs fall into subgroups in which specific patterns of cellular and immune functions may predominate.     

Correlation of the effect of feeding upon the cell cycle distribution profiles of MPC-11 cells with the relative appearance of [3H]-choline labeled material in microsomal subfractions and other cell fractions

Summary Cultures of mouse plasmacytoma cells (MPC-11) grown within the range 6–23 × 10⁵ cells/ml showed considerable variation in cell cycle distribution profiles and also differences with regard to relative amounts of microsomal subfractions. The variability of appearance of heavy rough (HR) and light rough (LR) microsomal subfractions was not merely due to differences in nutritional state of the culture. Cultures containing a high S/G₂ + M cell cycle distribution ratio showed a high content of HR microsomal membranes; as the S/G₂ + M ratio decreased, so too decreased the amount of HR material whilst the amount of LR microsomal membranes increased. The results indicate that there is a direct correlation between phase of cell cycle and both amount and relative distribution of rough microsomal membranes, the smooth fraction (S), however, remains relatively unchanged.     

Variations in Sulfhydryl, Disulfide, and Protein Content during Synchronous and Asynchronous Growth of HeLa Cells

The cellular contents of protein-bound and nonprotein sulfhydry (—SH) and disulfide (—SS—) groups were measured in both asynchronous and synchronous HeLa S3 cultures. About 90% of these groups are associated with proteins, the majority in the —SH form. The content of protein-bound groups, and hence the total content of —SH and —SS— groups (28 × 10^-15 moles/cell, or 1.1 × 10^-6 moles/g protein on average), changes in parallel with the protein content (which varies between 2 and 4 × 10^-10 g/cell) as asynchronous populations pass from the lag through the exponential to the stationary phase of growth. The concentration of nonprotein —SH groups, in contrast, increases 10-fold during lag phase and decreases in stationary phase; it follows the protein concentration closely during the exponential phase, at a level of about 2.8 × 10^-15 moles/cell. In synchronous cultures the protein content doubles during the cell cycle, possibly in an exponential fashion. The total —SH and —SS— content also doubles, but the rate of increase appears to fluctuate. The concentrations of the protein-bound groups show 2- to 3-fold fluctuations per unit protein: protein-bound —SH groups and mixed —SS— linkages rise to maxima while protein-bound —SS— groups fall to a minimum at the G₁/S transition, and fluctuations in these groups occur again during G₂. In addition, the protein-bound —SH concentration falls continuously during the S phase. The nonprotein —SH concentration undergoes the largest (relative) fluctuations, dropping from 4 × 10^-15moles/cell in early G₁ to about 0.4 × 10^-15 moles/cell (of standard protein content) at the end of G₁, and then rising to 30 times this value by the end of S.