Towards a further understanding of the growth-inhibiting action of oxygen deficiency. Evaluation of the effect of antimycin on proliferating Ehrlich ascites tumour cells

The effect of 1 microM antimycin on the proliferative properties, metabolism and basic cell composition of Ehrlich ascites tumour cells cultured in the second in vitro passage was studied. Continuous drug exposure of asynchronous cells caused rapid cessation of cell growth, characterized by the cell number and DNA, RNA and protein content of cultures. Cells cease to consume oxygen and enhance their glycolytic activity. Uptake of labelled thymidine into acid-insoluble material was far below that of the controls, whereas incorporation of labelled uridine exceeded that of controls, as was also observed with other inhibitors of the respiratory chain (sodium cyanide, 2-thenoyltrifluoroacetone, or anaerobiosis). The influence of antimycin on cells at different stages of the cell cycle was tested using cells enriched in either G1, S or G2 phase by centrifugal elutriation. DNA histograms (flow cytometry) and pulse-labelling index curves gave detailed insight into cell-cycle progression of antimycin-treated cells: G1 and early S cells remained stationary; G2 cells still passed from G2 into mitosis to remain subsequently in a non-growing state in G1; S cells were either slowed or halted. Supplementation of antimycin-containing cultures with exogenous pyrimidine nucleosides stimulated reprogression of G1 cells without changing their ATP content. The results of the current experiments are interpreted as supporting the concept that growth cessation of G1 cells under respiratory insufficiency is not predominantly caused by impairment of respiratory phosphorylation but may be the consequence of a lack of precursors for DNA and RNA synthesis.     

The respiration-linked limiting step of tumor cell transition from the non-cycling to the cycling state: Its inhibition by oxidizable substrates and its relationships to purine metabolism

The recruitment into the cycling state of resting Yoshida AH 130 hepatoma cells was studied with respect to its dependence on respiration in an experimental system wherein the overall energy requirement for this recruitment can be supplied by the glycolytic ATP. The G₁-S transition of these cells, unaffected by 2,4-dinitrophenol (DNP) at concentrations which uncouple the respiratory phosphorylation, is impaired either by blocking the electron flow to oxygen by antimycin A or by adding an excess of some oxidizable substrates, chiefly pyruvate and oxalacetate. An experimental analysis, focused on pyruvate activity, showed that the inhibition of cell recruitment into S is not related to the depressing effects of this substrate on aerobic glycolysis of tumor cells, nor is it modified by forcing, in the presence of DNP, pyruvate oxidation through the tricarboxylic acid cycle as well as the overall oxygen consumption. Addition of suitable concentrations of preformed purine bases (mainly adenine), completely removes the block of the G₁-S transition produced either by the excess of oxidizable substrates or by antimycin A. These findings indicate the existence of a respiration-linked step in purine metabolism, which restricts the above transition and is equally impaired by blocking the respiratory chain or by saturating it with an excess of reducing equivalents derived from unrelated oxidations. The inhibitory effects of pyruvate and antimycin A can be largely removed by the addition of folate and tetrahydrofolate, suggesting that the respiration-linked restriction point of tumor cell cycling involves the folate metabolism and its connections to purine synthesis.     

Selective inhibition of thymidine transport at low doses of the alkylating agents triethyleneiminobenzoquinone (Trenimon)

The alkylating antitumor agent triethyleneiminobenzoquinone (Trenimon) causes a rapid decrease in the incorporation of labeled thymidine into the DNA of Yoshida or Ehrlich ascites tumor cells. The effect is expressed 4 h after administration of 6 × 10⁻⁸ moles/kg of the drug to mice bearing Yoshida ascites tumors or of 6 × 10⁻⁷ moles/kg to Ehrlich ascites tumor-bearing animals, respectively. The reduced incorporation of labeled thymidine which is observed under these conditions is not due to an inhibition of DNA synthesis. DNA synthesis was measured by an isotope dilution assay after pulse-labeling with ³H-thymidine and by monitoring the increase in the total amount of DNA of the cell populations. The data demonstrate that DNA synthesis is not affected during the first 8 h after exposure to the drug. This conclusion is supported by cell kinetic measurements which indicate that the alkylating agent does not interfere with the progression of cells into the S phase, but exerts a block at the G 2 stage of the cell cycle. The reduced incorporation of thymidine into DNA is explained by a decreased transport of the nucleoside into the cells.     

Protein synthesis, ribosomal protein S6 phosphorylation in vitro and the effects of amiloride: SDS gel electrophoresis studies in the Yoshida ascites tumor (AH 130) grown in vivo

Cell-free cytosolic extracts from the Yoshida (AH 130) rat ascites hepatoma cell line, grown in vivo, showed high ribosomal protein S6 kinase activity in vitro, as measured by transfer of 32P to exogenous 40S rat liver ribosomal subunits, in both exponential growing and stationary phase cells. A significant decrease of protein synthesis (3H-leucine incorporation into total cell protein) was found to occur in cells reaching the stationary phase of growth, suggesting that S6 phosphorylation was not tightly coupled to the rate of the intraperitoneal cell growth and of protein synthesis in these tumor cells. When the cell-free cytosolic extracts were prepared from cells exposed to amiloride, at concentrations that inhibit the Na+/H+ exchange, a decrease of S6 kinase activity was observed only in exponential growing cells, suggesting the possibility of coupling of the Na+/H+ exchange with phosphorylation of intracellular proteins in these tumor cells. Actually, stationary phase cells showed unchanged S6 kinase activity under the same conditions, possibly due to the extremely low Na+/H+ exchange activity, previously demonstrated (Cell Biol. Int. Rep., 1985, 9, 1017-1025). The present experiments support the hypothesis that the regulation of protein synthesis is not tightly coupled to phosphorylation-dephosphorylation cycles, at least of ribosomal protein S6, in cells characterized by a rather uncontrolled growth such as the Yoshida (AH 130) rat ascites hepatoma. In this connection, an elevated degree of protein phosphorylation, such as that of the ribosomal protein S6, could be a general phenomenon of neoplastic transformation.     

Human diploid fibroblasts (HDF) can induce DNA synthesis in cycling HDF but not in quiescent HDF or senescent HDF

HeLa cells in S phase induce DNA synthesis in cycling cells, serum-deprived quiescent cells, and non-replicative senescent cells following cell fusion. In contrast normal human diploid fibroblasts (HDF) do not induce DNA synthesis in either quiescent cells or senescent cells. Instead, the replicative HDF nuclei are inhibited from entering S phase in heterokaryons formed with these two types of non-replicative cells. These differences in the inducing capabilities of normal HDF and HeLa cells raise the question whether normal HDF in S phase can induce DNA synthesis in cycling cells. This paper demonstrates that young HDF in S phase can induce DNA synthesis in cycling HDF. Thus, the hypothesis that initiation of DNA synthesis in cycling cells is positively controlled by inducer molecules appears to be valid for normal HDF as well as for transformed cells such as HeLa.     

Regulation of proliferating cell nuclear antigen during the cell cycle

The proliferating cell nuclear antigen (PCNA), also known as cyclin and DNA polymerase delta auxiliary factor, is present in reduced amounts in nongrowing cells and is synthesized at a greater rate in the S phase of growing cells. The recently discovered involvement of PCNA in DNA replication suggested that this pattern of expression functions to regulate DNA synthesis. We have investigated this possibility further by examining the synthesis, stability, and accumulation of PCNA in HeLa cells fractionated by centrifugal elutriation into nearly synchronous populations of cells at various positions in the cell cycle. In these fractionated cells we found that there is an increase in the rate of PCNA synthesis with a peak in early S phase of the cell cycle, but the magnitude of the increase is only 2-3-fold. This change reflects similar changes in the amount of PCNA mRNA. The fluctuating synthesis of PCNA maintains this protein at a roughly constant proportion of the total cell protein, although the amount doubles/cell in the cell cycle. Consistent with this observation, the stability of PCNA does not differ significantly from that of total cellular protein in synchronized HeLa cells. We also observed that a maximum of one-third of the total PCNA is tightly associated with the nucleus, presumably in replication complexes, at the peak of S phase. We conclude that the cyclic synthesis of PCNA in cycling HeLa cells maintains PCNA in excess of the amount involved directly in DNA replication and the amount of the protein neither fluctuates significantly with the cell cycle nor is limiting for DNA synthesis.     

Structuro-functional properties of the bacteria Bacillus brevis in relation to the accumulation of gramicidin S in cells

The culture of Bacillus brevis var. G-B R-form was grown in the presence of beta-phenyl-beta-alanine, the inhibitor of gramicidin S synthesis, is characterized by enhanced endogenous respiration and the DPI-reductase activity as compared to the culture synthezising antibiotic. The increased synthesis of the antibiotic in the region of the culture transition from the logarithmic growth phase to the linear one is associated with a decrease in the number of viable cells despite the fact that the culture on the whole does not die but continues to grow. The membranes prepared from young gramicidin S-free cells and from the cells enriched with the antibiotic possess identical electron micrograph images, IR spectra and protein sets as determined by polyacrylamide gel electrophoresis in a Na-DS system. However, in young cell membranes NADH and succinate dehydrogenase are insensitive to gramicidin S and only malate dehydrogenase is inhibited by this antibiotic. In aged cell membranes the activities of all mentioned dehydrogenases are suppressed. Malate dehydrogenase from young cells is weakly inhibited by thyrotrycin obtained from Bac. brevis ATCC 10068; succinate dehydrogenase is entirely insensitive to this antibiotic, while NADH-dehydrogenase is almost completely inhibited by it. The specificity of action on the respiratory chain of peptide antibiotics synthesized by the cells of one strain of Bac. brevis is suggestive of a possible regulatory role of these peptides in the metabolism of the producent. Hence the accumulation of gramicidin S which is adsorbed on the membrane and destroys the respiratory chain function to the cause of the low rate of oxygen uptake by the culture of Bac. brevis var. G-B R-form and of the low activities of DPI-reductases.     

Deficiency of joining of Okazaki-type fragments in absence of cellular protein synthesis.

The dependence of integration of newly formed DNA chain ( less than 10 S) into larger DNA on concomitant protein synthesis was studied in a special cellular system. Exponentially growing Ehrlich ascites tumor cells in vivo show decreasing rated and finally complete cessation of protein and DNA synthesis upon transfer into an isotonic but non-nutritive environment (Hanks' balanced salt solution). Both protein and DNA synthesis is stimulated in these cells for a period of 30 min when they are placed into fresh Hanks' balanced salt solution; however, stimulation of protein synthesis is completely prevented in Hanks' balanced salt solution containing cycloheximide. This system allowed us to investigate the formation and fate of newly formed DNA chains ( less than 10 S) in dependence of protein synthesis. Analysis of DNA produced in [3H]thymidine pulses showed that DNA chains smaller than 18 S were still formed during the phase of totally delayed protein synthesis and in the presence of cycloheximide, but they were not converted into DNA molecules sedimenting faster than 18 S under these conditions. Stimulation of protein synthesis for a period of 30 min allowed the short DNA pieces to be chased into larger DNA 30 min post stimulation of protein synthesis. The results clearly indicate that DNA chain growth, by sealing of DNA chains smaller than 18 S, is strongly dependent of concomitant protein synthesis. Direct chain elongation by addition of new deoxyribonucleotides is less dependent on concomitant cellular protein synthesis.     

Energy metabolism and ATP turnover time during the cell cycle of Ehrlich ascites tumour cells

The energy production in different parts of the cell cycle due to aerobic and aerobic glycolytic metabolism and ATP turnover time was estimated by measuring the oxygen consumption, lactate-pyruvate and ATP content of Ehrlich ascites tumour cells growing in vivo. Cell fractions of high purity from the various parts of the cell cycle were obtained by means of elutriator centrifuging. The total energy production for one cell cycle was estimated to be 19 × 10^?12 mol ATP, 60% of which was due to the aerobic metabolism. Whereas the total ATP production is unchanged during G1 a fairly exponential increase is found during the S and G2 + M phases. The total cellular ATP content increases from 12 fmol ATP at early G1 to 28 fmol ATP at G2 + M; this increase, however, is discontinuous and is most pronounced during G1 and during late S phase S phase/G2 + M. The ATP turnover time, as defined as the ratio between ATP content and ATP production, was found to increase significantly from 75 sec in early G1 to 120 sec in late G1 but was constantly 100 sec during the early, middle and late S phase as well as G2 + M. These variations indicate maximum energy-requiring processes during early G1 period of the cell cycle and are discussed in relation to K⁺Na⁺ flux and macromolecule synthesis.     

Expression and phosphorylation of the replication regulator protein geminin

It has been described that the replication regulator protein geminin is rapidly degraded at the end of mitosis and newly expressed at the beginning of the next S phase in the metazoan cell cycle. We have performed experiments to investigate the synthesis of geminin in cycling human HeLa cells. The levels of geminin-mRNA vary only modestly during the cell cycle with a 2-3-fold higher mRNA level at the G1/S phase transition, whereas newly synthesized geminin can only be detected in post-G1 phases. Surprisingly, geminin, once synthesized, does not remain stable, but is turned over during S phase with a half-life of 3-4h. We also show that geminin becomes phosphorylated as S phase proceeds and identify by MALDI mass spectrometry two specific major phosphorylation sites.     

Further characterization of the growth inhibitory effect of rotenone on in vitro cultured Ehrlich ascites tumour cells

Summary As an approach for a better understanding of the mode of action of rotenone on mammalian cells we have studied the proliferation properties, metabolism and basic cell composition of Ehrlich ascites tumour cells cultured in vitro in the presence of 2,5 µM rotenone and after removal of the inhibitor.Experiments on asynchronous cells showed a rapid cessation of cell division accompanied by increased glycolytic rate, reduced oxygen consumption, moderate increase in DNA content and a fair increase in protein and RNA content of the cultures. DNA histograms obtained by flow-cytometry revealed an accumulation of cells in the G2 and M phase of the cell cycle. Electron micrographs taken after a 24 h treatment of cells illustrated the formation of giant mitochondria and fragmented nuclei.In order to elucidate the dual effect of rotenone — inhibition of mitochondrial energy metabolism and of mitotic processes — the influence on cells of rotenone at different stages of the cell cycle was tested using Ehrlich ascites tumour cells enriched in G1, S and G2 by centrifugal elutriation. DNA histograms and [³H]thymidine labelling index curves of cells from the different fractions cultured in the presence of 2,5 AM rotenone indicated that in addition to the observed accumulation in G2 and mitotic arrest of cells, the cell cycle progression is delayed in G1 phase. This may be explained by an effect of the inhibitor on the respiratory chain. S phase cells seemed to continue the cycle for several hours at a rate comparable to that of controls.Recultivation experiments on rotenone-treated asynchronous cells in inhibitor-free medium confirmed that some cells reinitiate DNA synthesis without preceeding cell division.Thus it must be concluded that cells at all stages of the cycle are affected by rotenone, but the impairment of cellular metabolism becomes manifest and lethal as soon as the acute block at mitosis is abolished and cells reenter the cycle.Abbreviations EAT cells Ehrlich ascites tumour cells - Hanks' solution Hanks' balanced salt solution - Hepes 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid     

Effect of ATP analogs of DNA synthesis in isolated nuclei

Optimal synthesis of DNA in Ehrlich ascites cell nuclei is shown to be dependent upon the presence of both ATP and ADP. ATP can be replaced only by dATP. An ATP regenerating system is less effective than ATP alone or ATP in combination with ADP. ATP does not stimulate DNA synthesis primarily by maintenance of deoxyribonucleotide triphosphate levels. When the inhibition of DNA synthesis by high ATP levels is taken into account, the ATP analogs adenosine 5'-(alpha,beta-methylene)triphosphate, adenosine 5'-(beta, gamma-methylene)-triphosphate, and adenosine 5'-(beta, gamma-imino)triphosphate can neither substitute for ATP nor inhibit the ATP stimulation of DNA synthesis. Adenosine 5'-(3-thio)triphosphate, however, is a competitive inhibitor of DNA synthesis.     

Effect of cycloheximide, 2,4-dinitrophenol and papaverine on ribosomal RNA synthesis in Ehrlich ascitic carcinoma cells]

The influence of 2,4-dinitrophenol (DNP), papaverine and cycloheximide on RNA synthesis in Ehrlich ascites tumour cells has been investigated. All above mentioned agents inhibit selectively synthesis of high-molecular rRNA precursor, when the cell population density is 3.10(7)--5.10(7) per 1 ml of suspension. When the density of cells decreases as far as 1.10(6) cells per 1 ml. the rRNA synthesis loses the sensitivity to all these agents. The effects of both cycloheximide on the protein synthesis and DNP on ATP level do not depend on the cell population density in suspension. It is suggested that either with a decrease of cell population density the protein synthesis and ATP level cease playing the role of a rate-limiting factor in the rRNA synthesis, or the influence of agents studied is realized by means of their interaction with other cell system.     

Cellular Ras and Cyclin D1 Are Required during Different Cell Cycle Periods in Cycling NIH 3T3 Cells

Novel techniques were used to determine when in the cell cycle of proliferating NIH 3T3 cells cellular Ras and cyclin D1 are required. For comparison, in quiescent cells, all four of the inhibitors of cell cycle progression tested (anti-Ras, anti-cyclin D1, serum removal, and cycloheximide) became ineffective at essentially the same point in G1 phase, approximately 4 h prior to the beginning of DNA synthesis. To extend these studies to cycling cells, a time-lapse approach was used to determine the approximate cell cycle position of individual cells in an asynchronous culture at the time of inhibitor treatment and then to determine the effects of the inhibitor upon recipient cells. With this approach, anti-Ras antibody efficiently inhibited entry into S phase only when introduced into cells prior to the preceding mitosis, several hours before the beginning of S phase. Anti-cyclin D1, on the other hand, was an efficient inhibitor when introduced up until just before the initiation of DNA synthesis. Cycloheximide treatment, like anti-cyclin D1 microinjection, was inhibitory throughout G1 phase (which lasts a total of 4 to 5 h in these cells). Finally, serum removal blocked entry into S phase only during the first hour following mitosis. Kinetic analysis and a novel dual-labeling technique were used to confirm the differences in cell cycle requirements for Ras, cyclin D1, and cycloheximide. These studies demonstrate a fundamental difference in mitogenic signal transduction between quiescent and cycling NIH 3T3 cells and reveal a sequence of signaling events required for cell cycle progression in proliferating NIH 3T3 cells.     

Uridylation of U6 RNA in a nuclear extract in Ehrlich ascites tumor cells

The uridylation of U6 RNA in a nuclear extract of Ehrlich ascites tumor cells was examined. This reaction required ATP or GTP, although these nucleotides were not incorporated into U6 RNA itself. ATP and GTP could be replaced by their nonhydrolyzable analogues ATP gamma S and GTP gamma S. Therefore, hydrolysis of ATP or GTP is not necessary for the uridylation of U6 RNA, indicating that these nucleotides are effectors of this reaction. By chromatographies of a nuclear extract of Ehrlich ascites tumor cells on phosphocellulose and DEAE-cellulose, U6 RNA could be separated from an enzyme adding a uridine residue(s) to this RNA.     

Cell cycle related [3H]thymidine uptake and its significance for the incorporation into DNA

Cellular uptake of [3H]thymidine [( 3H]TdR) and incorporation into DNA of Ehrlich ascites tumour cells were studied in relation to the cell cycle by measuring the activity in the acid-soluble and insoluble parts of the cell material. Cells were synchronized at various stages of the cell cycle using centrifugal elutriation. The degree of synchrony of the various cell fractions was measured by flow-cytofluorometric DNA analysis. From the cellular uptake, the TdR triphosphate (dTTP) concentration of a mean cell in an unseparated cell population was calculated to be 20 X 10(-18) mol/cell. The pool activity of G1 cells was unmeasurable but rose to maximum values at the border of the G1-S phase. It decreased again during G2. The [3H]TdR incorporation into DNA was low during early S phase, reached a maximum value at two-thirds of the S phase and decreased again during late S phase. These changes in DNA synthesis were not due to changes in the dTTP pool being a limiting factor. During maximum DNA synthesis, 10% X min-1 of the dTTP pool was utilized, at which time the pool size also decreased by about 30%. Changes in pool size during the cell cycle have to be taken into account when the results of incorporation of radioactive TdR into DNA are discussed.     

A cytoplasmic cell cycle controls the activity of a K+ channel in pre-implantation mouse embryos.

We previously have reported that the activity of a 240 pS K+ channel varies during the cell cycle in pre-implantation mouse embryos. In the present study, we show that: (i) the cycling of channel activity is not prevented by inhibiting protein synthesis and hence does not involve cyclin-dependent kinase 1 (cdk1)-cyclin B; and (ii) the cycling of channel activity continues in anucleate zygote fragments with a time course similar to that observed in nucleate fragments. We further demonstrate that: (i) persistent activation of the K+ channel in one-cell embryos arrested in metaphase requires the maintenance of an active cdk1-cyclin B complex; and (ii) both DNA synthesis inhibition with aphidicolin and DNA damage produced by mitomycin C prevent the down-regulation of the channel at the start of S phase by a mechanism that requires tyrosine kinase activation. Thus, the 240 pS K+ channel in these cells is controlled by a previously unsuspected cytoplasmic clock that functions independently of the well-known clock controlling the chromosomal cell cycle, but can interact with it.     

Poly(A) polymerase activity during cell cycle and erythropoietic differentiation in erythroleukemic mouse spleen cells.

Poly(A) polymerase activity was studied in lysates of cultured murine erythroleukemic cells (Friend cells). Incorporation of ATP into acid-precipitable products is dependendent on the presence of Mn2+ or Mg2+ and of an RNA primer. The reaction is specific for ATP as the substrate (KM=290 290 micron, it is not inhibited by actinomycin D and only slightly interferred with by ethidium bromide. Cordycepin 5'-triphosphate and sodium pyrophosphate inhibit the enzyme activity. The chain length of the products of the reaction is dependent on the primer concentration and reaches up to 30 nucleotides. Poly(A) polymerase activity is low in resting (G1 phase) cells 75 nmol ATP incorporated/h per 10(6) cells) and increases to a level about twice as high in early S phase of the cell cycle. A possible model for regulation of enzyme activity is discussed. Polymerase activity in the early phase of erythropoietic differentiation of the cells induced by butyric acid does not show any difference in comparison to untreated controls. A decrease in enzyme activity to levels characteristic for cells in G1 phase accompanies shutdown of cell growth in the course of the ongoing differentiation. Analysis of the DNA content of the cells revealed that erythropoietic differentiation of Friend cells induced by butyric acid is characterized by arrest of the cells in G1 phase of the cell cycle. Poly(A) polymerase activity in erythroleukemic cells is thus controlled only by the phase of the cell cycle; it is not affected by changes in gene expression during erythroid differentiation.     

Altered proteoglycan synthesis via the false acceptor pathway can be dissociated from beta-D-xyloside inhibition of proliferation.

beta-D-Xylosides have been used to perturb proteoglycan (PG) synthesis to elucidate the function of PGs in a number of cellular processes, including proliferation, migration, and differentiation. This study was designed to examine whether specific xylosides affect the proliferation of several different cell types and, if so, whether this effect is dependent on altered PG synthesis via the false acceptor pathway. Both methylumbelliferyl beta-D-xylopyranoside and p-nitrophenyl beta-D-xylopyranoside (PNP beta-xyloside) inhibit cell proliferation and modulate PG synthesis; however, the alpha form of PNP xyloside which does not perturb PG synthesis inhibits the proliferation of cultured cells on a molar basis equally as well as the beta form. Conversely, beta-methyl xylopyranoside stimulates the synthesis of free glycosaminoglycan chains equally as well as PNP beta-xyloside and yet has no measurable effect on cell proliferation at comparable doses, indicating that cells can grow normally while experiencing disruption of their proteoglycan metabolism. At doses ranging from 0.5 to 5 mM, PNP beta-xyloside arrests cells in the G1 phase of the cell cycle at the same time point as serum starvation. It also delays the exist of cycling cells from the S phase. This treatment is not cytotoxic and is rapidly reversed by the replacement of PNP beta-xyloside containing medium with control medium. Dimethyl sulfoxide, the most commonly used solvent for beta-xyloside in proteoglycan studies, potentiates the inhibitory effect of PNP beta-xyloside on cell proliferation. These results indicate that the perturbation of PG synthesis via the false acceptor pathway can be uncoupled from control of cell proliferation.