Inhibition of growth and of thymidine incorporation into DNA in Tetrahymena by chlorpromazine, pimozide and penfluridol

The antipsychotic drugs chlorpromazine, pimozide, and penfluridol caused a 50% inhibition of growth of Tetrahymena at concentrations of 4.5, 5.5, and 1.5 microM, respectively. The degree of growth inhibition was dependent on the concentration of cells; higher drug concentrations were needed to produce inhibition of denser cell cultures. Binding studies with penfluridol showed that 50% growth inhibition resulted when approximately 50 mumoles of drug were bound per 10(6) cells. A 20-min preincubation of cells with chlorpromazine (14.7 microM) inhibited DNA synthesis by 46%, and with penfluridol (4 microM) DNA synthesis was inhibited by 27%. The incorporation of labeled thymidine into the thymidine triphosphate pool was inhibited by chlorpromazine but not by penfluridol, indicating that the drugs produce their growth inhibitory effects by different mechanisms. TDP kinase activity was demonstrated in a particle-free fraction of the cells. Its enzymatic activity was not affected by added chlorpromazine, penfluridol, or calmodulin, suggesting that inhibition of DNA synthesis by these drugs may be a consequence of growth inhibition.     

Inhibition by phenothiazine antipsychotic drugs of calcium-dependent phosphorylation of cerebral cortex proteins regulated by phospholipid or calmodulin

Phospholipid-sensitive and calmodulin-sensitive Ca²⁺-dependent phosphorylation of a number of endogenous proteins in the soluble and particulate fractions of the rat cerebral cortex was inhibited by phenothiazine antipsychotic drugs. The mean IC₅₀ values (concentrations causing 50% inhibition of phosphorylation) for trifluoperazine, chlorpromazine and fluphenazine were 16, 24 and 27 μM, respectively. Dibucaine, a local anesthetic drug, was much less effective. It appears that these neuroleptic agents may be useful tools for the study of Ca²⁺-dependent protein phosphorylating systems regulated by either phospholipid or calmodulin in the brain.     

Comparison of the Selectivity of Anti-Varicella-Zoster Virus Nucleoside Analogues

We compared the selectivity of six anti-varicella-zoster virus (VZV) drugs, which are clinically available or of which clinical efficacy for the treatment of VZV infections has been reported. Sorivudine (BV-araU) had the most potent anti-VZV effect in the plaque inhibition assay, followed by brivudine (BVDU) and 5-propynyl-arabinofuranosyluracil (Pry-araU). All test compounds, except vidarabine (AraA), had only a very weak effect on human embryonic lung cell growth. The selectivity indexes (ID₅₀ for cell growth/ED₅₀ for VZV plaque inhibition) of BV-araU, BVDU, and Pry-araU were > 1,000,000, 20,000, and > 10,000, respectively, while those of acyclovir and penciclovir ranged from 600 to 800. AraA was much less selective than any of the other drugs tested. We measured the amount of pH] thymidine incorporated into the acid-insoluble fraction of VZV-infected cells to determine the ability of these drugs to selectively inhibit viral DNA synthesis. [³H]Thymidine incorporation was markedly inhibited by all anti-VZV compounds, except BVDU. Treatment of infected cells with drugs from 32 to 38 hr after infection inhibited the DNA synthesis to the same extent as VZV plaque formation, except that AraA inhibited the DNA synthesis at a lower dose than for VZV plaque formation. DNA synthesis in non-infected growing cells was inhibited to the same extent as cell growth. A particularly high selectivity index for the inhibition of DNA synthesis was noted for BV-araU, which was defined as the ratio of inhibitions of DNA synthesis in VZV-infected and non-infected. The highest selectivity indexes were recorded for BV-araU > Pry-araU > acyclovir ≥ penciclovir > AraA.     

Demonstration of a late amiloride-sensitive event as a necessary step in initiation of DNA synthesis by thrombin

Amiloride, a Na⁺ influx inhibitor, has been shown to inhibit initiation of DNA synthesis by thrombin in mouse embryo fibroblast-like cells. Long exoosures (24 hr) to high concentrations of amiloride inhibited incorporation of thymidine into the DNA of both thrombin-stimulated and nonstimulated cells, suggesting that this inhibition might not be specific for thrombin-initiated DNA synthesis. Fluorescence microscopy and spectrofluorimetry showed that amiloride was internalized with an apparent mitochondrial association and that the internalized amiloride was readily released from the cells after removing amiloride from the medium. Based on this reversibility, cells were exposed to amiloride for short periods of time during thrombin treatment to determine the temporal relationship between any amiloride-sensitive event(s) and initiation of DNA synthesis. The presence of amiloride (100 μM) during a 12-hr exposure to thrombin did not block thrombin-initiated DNA synthesis or cell division but did delay the onset of DNA synthesis and the peak of thymidine incorporation into DNA by approximately 3 hr, suggesting that early initiation events might proceed in the presence of amiloride. ⁸⁶Rb⁺ transport studies demonstrated that in this system ouabain-sensitive K⁺ uptake via the Na, K-ATPase was stimulated by thrombin during both an early and a late period. This stimulation was amiloride-sensitive under the same conditions used for growth experiments, suggesting that amiloride was inhibiting thrombin-stimulated Na⁺ transport in this system. Additional experiments showed that exposing cells to amiloride only during the first 8 hr after thrombin addition did not inhibit initiation. The presence of amiloride from 8–12 hr after thrombin addition maximally inhibited thrombin-stimulated DNA synthesis. Together these results demonstrate that amiloride inhibits thrombin-initiated DNA synthesis not by inhibiting an early event occurring during the first 8 hr, but rather by inhibiting some later event 8–12 hr after thrombin addition.     

The response of normal and polyoma virus-transformed BHK-21 cells to exogenous purines

Adenosine (10 μM) stimulates the initial growth rate of BHK/21 cells seeded at low but not high density in monolayer culture; it does not affect final cell density or permit growth in agar. In labelling experiments With tritiated thymidine, adenosine also increases the response of quiescent cells to low concentrations of serum. Dialysis of serum to remove oxypurines only marginally reduces its effect on quiescent cell labelling or growth, indicating that BHK/21 cells are able to synthesise purines. The response of quiescent cells to 5% serum is inhibited by high MW (2 × 10⁶) dextran sulphate at 2 μg per milliliter. Low MW dextran sulphate (30,000) and heparin at 20 μg per milliliter produce the same effect. Exogenous adenosine (10 μM) prevents this inhibition. Many other purine derivatives replace adenosine for all the above activities but xanthine is completely inactive in all. It, therefore, appears that nucleotide synthesis is a necessary function of these compounds. The growth of cells of a polyoma-virus-transformed BHK/21 line in monolayer is not stimulated by exogenous purine, though their colony-forming ability in agar is increased five-fold. The stimulating effects of exogenous purines on normal BHK/21 cells and the absolute requirement for them in the presence of polyanions is discussed in relation to possible mechanisms of growth control.     

DNA repair and replication in human fibroblasts treated with (±)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

DNA repair and replication were examined in diploid human fibroblasts after treatment with (±)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I). Unscheduled DNA synthesis exhibited a linear response to BPDE-I concentrations up to 1.5 μM and a saturation plateau after higher concentrations. Maximal unscheduled DNA synthesis was observed in the first hour after treatment with synthesis diminishing progressively thereafter. Half-maximal unscheduled DNA synthesis was seen within 4–6 h after treatment with 0.7 μM BPDE-I. DNA replication was inhibited by BPDE-I in a dose- and time-dependent fashion. The mechanisms of this inhibition were characterized by velocity sedimentation of pulse-labeled nascent DNA in alkaline sucrose gradients. Very low concentrations of BPDE-I (0.03 and 0.07 μM) were found to inhibit replicon initiation by up to 50% within 30–60 min after treatment. Recovery of initiation following these low concentrations was evident within 3 h after treatment. Higher concentrations of carcinogen inhibited DNA synthesis in active replicons. This effect was manifested by a reduction in incorporation of precursor into replication intermediates of greater than 1·10⁷ Da with the concurrent production of abnormally small nascent DNA. When viewed 45 min after treatment with 0.17 μM BPDE-I the combination of these two effects partially masked the inhibition of replicon initiation. However, even after treatment with 0.33 μM BPDE-I an effect on initiation was evident. These results reveal a pattern of response to BPDE-I that is quite similar to that produced by 254 nm radiation.     

Prostaglandin production by mouse fibrosarcoma cells in culture: inhibition by indomethacin and aspirin

Five clonal strains of mouse tumor cells (HSDM₁) synthesize and secrete large quantities (0.70-2.0 μg/mg cell protein/24 hr) of prostaglandin E₂. Five lines of control cells did not synthesize significant amounts of prostaglandins. HSDM₁ cells produce prostaglandin E₂ during both the logarithmic and stationary phases of the cell growth cycle. Prostaglandin production was inhibited by aspirin-like drugs; for example, 50% inhibition was obtained with as little as 3 × 10⁻⁹ M indomethacin. We conclude that the HSDM₁ cell system will serve as a useful model system to study prostaglandin synthesis and secretion.     

Effect of arabinosyl cytosine on the level of DNA polymerase and thymidine kinase activity in PHA-stimulated human tonsillar lymphocytes

Summary The effect of arabinosyl cytosine (ara-C) was studied on the uptake, phosphorylation and incorporation of ³H-thymidine in human tonsillar lymphocyte cultures is described along with its effect on the level of DNA polymerase and thymidine kinase activities induced by phytohaemagglutinin (PHA). Freshly isolated tonsillar lymphocytes are stimulated cells with a remarkably high activity of DNA polymerase a and thymidine kinase. During in vitro culture, these stimulated cells are transformed to the resting state with low DNA polymerase and thymidine kinase activity. However, a new DNA synthesising cycle can be induced by PHA with maximum at 48 h.10^–6 M ara-C inhibited the incorporation of ³H-thymidine by 90–95%. This inhibition may be reversed by rinsing the cells. The inhibition of the transport of ³H-thymidine seems to be only a consequence of the inhibitory effect of ara-C on the DNA polymerisation reaction, because at 10 °C, where DNA synthesis was arrested, ara-C does not influence the uptake and the phosphorylation of ³H-thymidine.Ara-C (10^–6 M) abolished also the PHA induced elevation of DNA polymerase a and thymidine kinase activities without influencing protein synthesis of the cell. This supports a coordinated regulation mechanism between DNA synthesis and the synthesis of enzymes involved in DNA replication.     

Daunomycin disrupts nuclear assembly and the coordinate initiation of DNA replication in Xenopus egg extracts

We have used Xenopus egg extracts to investigate the effects of the antitumor drug daunomycin on DNA replication in vitro. Xenopus sperm nuclei replicated nearly synchronously in our egg extracts, thereby allowing us to determine the effects of the drug on both replication initiation and elongation. Titration experiments demonstrated that daunomycin effectively inhibited replication in the extract, with 50% inhibition at a total drug concentration of 2.7 μM. However, a high concentration of daunomycin 150 μM) also inhibited nuclear envelope assembly, a prerequisite for the initiation of replication in this system. Therefore, to bypass the effects of daunomycin on nuclear envelope assembly, sperm nuclei were preassembled in extract prior to drug addition. Initiation of replication in preassembled nuclei was also inhibited by daunomycin, with 50% inhibition at a drug concentration of 3.6 μM. At low drug concentrations, where replication did occur, the synchrony of initiations within individual nuclei was lost. This drug-induced disruption of initiation events may provide important clues regarding the mechanism(s) by which these events are coordinated in eukaryotic cells. Daunomycin also inhibited replication elongation in preassembled, preinitiated nuclei. However, the concentration of drug required for 50% inhibition of elongation was nearly fourfold higher than that required for inhibition of initiation. Taken together, these data demonstrate that Xenopus egg extract can be used to investigate the effects of DNA-binding antitumor drugs on a number of interrelated cellular processes, many of which are less tractable in whole cell systems. J. Cell. Biochem. 64:476–491. © 1997 Wiley-Liss, Inc.     

Approach to the mechanism of Zajdela's hepatoma cell growth inhibition induced by concanavalin A and phytohaemagglutinin

Cell growth of tumour ascites cell was inhibited by concanavalin A, phytohaemagglutinin and Ricinus lectin at 2–100 μg/ml. As expected, the Ricinus lectin inhibited the protein synthesis estimated by leucine incorporation and decreased thymidine incorporation, whereas concanavalin A and phytohaemagglutinin stimulate the uptake and the incorporation of both leucine and thymidine, and thus, synthesis of protein and DNA. Theses results suggest that different mechanisms are involved in the hepatoma cell growth inhibition by the lectins. This difference was not related to the kinetic characteristics of the lectin interactions with the cells whihc represent a first and necessary step. It was showed that concanavalin A and phytohaemagglutinin as well as chloroquine inhibited the ¹⁴C-labelled asialofetuin degradation. We can conclude that Ricinus lectic present a toxic effect whereas both concanavalin A and phytohaemagglutinin show an anti-protease activity.     

Inhibition of mammalian DNA replication by dichlorobenzimidazole riboside

We have investigated the effect of the nucleoside analogue 5,6-dichloro-1-β- -ribofuranosyl-benzimidazole (DRB) on DNA synthesis in the L-929 line of mouse fibroblasts. Earlier studies have shown that this compound selectively inhibits the synthesis of a major fraction of nuclear heterogeneous RNA (hnRNA). At 60–75 μM, DRB decreases the rate of nuclear hnRNA synthesis by two-thirds and prevents the appearance in the cytoplasm of almost all poly(A)-containing messenger RNA (mRNA). At similar dose levels, DRB inhibits the overall rate of DNA synthesis as measured by thymidine incorporation by only 20% (after correction for decreased thymidine uptake into total cellular material). Analyses of density gradients of BUdR and [³H]thymidine-substituted DNA and of autoradiograms of extended fibers of [³H]thymidine-labeled DNA confirm this inhibition and suggest that DRB acts on DNA solely by reducing the rate of replication fork movement. The apparent size of replication units is unchanged by drug treatment. DRB also inhibits the transport of thymidine into cells. As determined by kinetic studies of thymidine uptake, transport is inhibited competitively by drug treatment. Several other aspects of thymidine metabolism are not affected by DRB. It does not alter the size of the total dTTP pool, and there is no breakdown of template DNA in DRB-treated cells.     

Deoxyribonucleoside triphosphate pools and differential thymidine sensitivities of cultured mouse lymphoma and myeloma cells

The effects of various concentrations of thymidine on DNA synthesis and deoxyribonucleoside triphosphate contents of a highly thymidine-sensitive cultured mouse lymphoma cell line (WEHI-7) and a relatively resistant mouse myeloma cell line (HPC-108) have been studied by 32P-labelling techniques. DNA synthesis in the myeloma cells was inhibited by thymidine at concentrations of 10(-3) M or greater, while DNA synthesis in the lymphoma cells was inhibited by concentrations 30-fold lower, consistent with the 25-fold difference between the two cell lines in sensitivity to growth inhibition by thymidine. Thymidine caused marked elevation of the dTTP and dGTP pools, slight elevation or no change in the dATP pool and a marked decrease in the dCTP pool in cells of both lines. The greater resistance of HPC-108 cells to thymidine inhibition was related to the finding that they normally contained a much higher concentration of dCTP than did the WEHI-7 cells. Pool size measurements on thymidine-treated (10(-4) M) cells of an additional seven sensitive lymphoma and six relatively resistant myeloma cell lines indicated that in all 15 lines studied, with one exception, a critical concentration of dCTP of about 32 nmol per ml of cell volume was required for the maintenance of normal rates of DNA synthesis. The dCTP content found normally in the lymphoma cells was only a little above this concentration. Amongst the myeloma lines, three contained similarly low levels of dCTP, but were more resistant to thymidine inhibition probably because of their inefficient production of dTTP from thymidine. Cells of the other four myeloma lines (including HPC-108) normally contained much higher dCTP concentrations. The mechanism of thymidine action was explained by reference to the known allosteric properties of ribonucleotide reductase.     

The Action of Mercaptoethanol on Cell Division in Synchronized Tetrahymena1

SYNOPSIS. Action of mercaptoethanol (ME) on cell division and macromolecular synthesis was examined in Tetrahymena synchronized for division. Cells continuously exposed to increasingly higher concentrations of ME divided with progressively longer division delays showing a dosage-dependent response to the agent. Division was blocked in 2 × 10^?2 M ME. Many cells cytolyzed in high concentrations of ME (4 × 10^?2 M); others became spherical and motility decreased. Non-delaying concentrations of ME (2 × 10^?2 M) had little or no effect on protein synthesis but decreased DNA and RNA synthesis 10 and 35%, respectively. Blocking concentrations inhibited incorporation of phenylalanine, thymidine and uridine 35, 60, and 85%, respectively. It is suggested that the mode of action of ME is mediated thru inhibition of macromolecular synthesis essential for cell division and thru inhibition of formation of disulfide bridges between protein subunits.     

Inhibition of nucleic acid synthesis in leukemia 1210 cells by antimetabolites of coenzyme Q10

Thirteen diversified antimetabolites of coenzyme Q₁₀ which have antitumor activity $\text{in vivo}$ were tested for inhibition of uptake of tritiated thymidine and uridine into DNA and RNA, respectively, of L1210 cells grown in tissue culture. Eight of these antimetabolites have inhibitory activities of the same order of magnitude as the used anticancer drugs, rubidazone and ellipticine. 5-ω-Phenylpropylmercapto-2,3-dimethoxy-1,4-benzoquinone was particularly potent to inhibit nucleic acid synthesis; ED₅₀ for DNA = 2.1 μM and ED₅₀ for RNA = 4.0 μM.     

A ROLE FOR MICROTUBULES IN THE REGULATION OF 3-HYDROXY-3-METHYLGLUTARYL COENZYME A REDUCTASE AND CHOLESTEROL BIOSYNTHESIS IN CULTURED GLIAL CELLS 1

—Cultured C-6 glial cells were utilized to evaluate the effect of the antimicrotubular drug, Colcemid, on 3-hydroxy-3-melhylglutaryl coenzyme A (HMG-CoA) reductase and cholesterol synthesis in cultured C-6 glial cells. The data indicate that Colcemid causes a marked inhibition of cholesterol synthesis (from [¹⁴C]acetate or ³H₂O) in these cells. A concentration of 0.5 μM led to a 50% lower rate of synthesis after 2 h and an 80–85% lower rate after 12 h or longer. That the effect of Colcemid is mediated at the level of HMG-CoA reductase was shown by defining closely coordinate temporal and quantitative changes in the activity of this enzyme under identical conditions. No comparable change in cell growth or in total protein synthesis accompanied the effect of Colcemid. The drug did lead to a decrease in the rate of DNA synthesis (from [³H]thymidine) but this effect was preceded by the decrease in the rate of cholesterol synthesis. Marked changes in glial cell shape were induced by exposure to Colcemid, and the temporal and quantitative aspects of these changes appeared to closely parallel the effects on reductase activily and cholesterol synthesis. The dala suggest that microtubules are involved in the regulation of HMG-CoA reductase and cholesterol synthesis in mammalian cells and that there are important interrelations between microtubules, glial differentiation and cholesterol synthesis.     

Evidence that microtubule depolymerization early in the cell cycle is sufficient to initiate DNA synthesis

Microtubule disrupting drugs initiated DNA synthesis in serum-free cultures of nonproliferating fibroblast-like cells. The addition of colchicine to chick, mouse and human fibroblasts in serum-free medium stimulated thymidine incorporation at least twofold, with a half-maximal concentration of 1 X 10(-7) M. This stimulation represented up to 75% of the maximal stimulation by thrombin and was paralleled by an increase in the percentage of labeled nuclei. Other microtubule disrupting drugs showed similar stimulation, whereas lumicolchicine had no effect. Indirect immunofluorescent staining of tubulin showed a correlation between microtubule depolymerization and initiation of DNA synthesis by these drugs. A 2 hr treatment with 10(-6) M colchicine caused complete disruption of the microtubular network and stimulated thymidine incorporation (measured 28 hr later) to an even greater extent than continuous colchicine exposure. A similar 2 hr exposure to 10(-6) M colcemid also stimulated thymidine incorporation and led to a 50% increase in cell number. Taxol, a drug which stabilizes cytoplasmic microtubules, blocks initiation of DNA synthesis by colchicine, indicating that microtubule depolymerization is necessary for this initiation. To determine if microtubule depolymerization is involved in stimulation of DNA synthesis by other growth factors, highly purified human thrombin was added to cells with or without colchicine. In no case did colchicine plus thrombin increase DNA synthesis above that of the maximal stimulation by thrombin alone. Furthermore, pretreatment of cultures with taxol (5 micrograms/ml) inhibited approximately 30% of the stimulation of thymidine incorporation by thrombin. Together, these studies demonstrate that microtubule depolymerization is sufficient to initiate both DNA synthesis and events leading to cell division and suggest that microtubule depolymerization may be a required step in initiation of cell proliferation by growth factors such as highly purified human thrombin.     

Reduced inhibition of DNA synthesis and G2 arrest during the cell cycle of resistant HeLa cells in response tocis-diamminedichloroplatinum

The influence of cisplatin, an anticancer agent, on DNA synthesis and cell cycle progression of a cisplatin-resistant cell line was investigated. Cell cycle analysis using flow cytometry showed that cytotoxic concentrations of cisplatin caused a transient inhibition of parental HeLa cells at S phase, followed by accumulation at G₂ phase. In contrast, the resistant cells progressed through the cell cycle without being affected by the same treatment. However, cell cycle distributions were the same in the resistant and the parental cells at IC₅₀, the drug concentration inhibiting cell growth by 50%. Studies using a [³H]thymidine incorporation technique also demonstrated a transient inhibition of DNA synthesis in HeLa cells by cisplatin; such inhibition was greatly reduced in the resistant cells. These data argue for the hypothesis that the inhibition of DNA synthesis is important in determining cisplatin-induced cytotoxicity. In addition, the accumulation of cells at G₀/G₁ by serum starvation was not effective in the resistant cells compared to the parental cells, suggesting that the control of cell cycle exiting is also altered in the resistant cells. Taken together, these results support the notion that alterations in cell cycle control, in particular G₂ arrest, are important in determining the sensitivity or resistance of mammalian cells to cisplatin and may have a role in clinical protocols.     

Effects of adenine arabinoside on lymphocytes infected with Epstein-Barr virus.

Low concentrations of adenine arabinoside inhibited growth of two Epstein-Barr virus producer cell lines in culture, while not significantly affecting a nonproducer cell line and a B-cell-negative line. These observations were extended to include freshly infected cells. Mitogen-stimulated human umbilical cord blood lymphocytes were unaffected by the drug at concentration levels that inhibited [3H]thymidine incorporation into the DNA of Epstein-Barr virus-stimulated cells. DNA synthesis in Epstein-Barr virus-superinfected Raji cells was also adversely affected by adenine arabinoside. However, these same low concentrations of adenine arabinoside in the triphosphate form produced less effect on DNA synthesis in nuclear systems and DNA polymerase assays than on growth or DNA synthesis in whole cells. Therefore the effects reported here of low concentrations of the drug on whole cells may be only in part related to DNA polymerase inhibition. The work reported here suggests that adenine arabinoside has multiple sites of action in infected cells.     

Deoxyribonucleoside triphosphate metabolism and the mammalian cell cycle. Effects of hydroxyurea on mutant and wild-type mouse S49 T-lymphoma cells

DNA precursor synthesis can be blocked specifically by the drug hydroxyurea (HU) which has therefore been used for anticancer therapy. High concentrations of HU, however, affect other processes than DNA synthesis; nevertheless, most studies on the biological action of HU have been made with concentrations at least one order of magnitude higher than those needed for cell-growth inhibition. In this study we characterized the effects of low concentrations of HU (i.e. concentrations leading to 50% inhibition of cell growth in 72 h) on cell cycle kinetics and nucleotide pools in mouse S49 cells with various defined alterations in DNA precursor synthesis. The effect of 50 microM HU on deoxyribonucleoside triphosphate pools was a 2-3-fold decrease in the dATP and dGTP pools, with no change in the dCTP pool and a certain increase in the dTTP pool. Addition of deoxycytidine or thymidine led to a partial reversal of the growth inhibition and cell-cycle perturbation caused by HU, and was accompanied by an increased level of the deoxyribonucleoside triphosphates. Addition of purine deoxyribonucleoside gave no protection, indicating that salvage of these nucleosides could not supply precursors for DNA synthesis in T-lymphoma cells. We observed a higher sensitivity to HU of cells lacking purine nucleoside phosphorylase or with a ribonucleotide reductase with altered allosteric regulation. Cells lacking thymidine kinase or deoxycytidine kinase were just as sensitive as wild-type cells.     

Dissociation by cytochalasin B of movement, DNA synthesis and transport in 3T3 cells.

Cytochalasin B was used as a tool to study the inter-relationships between cell movement, the reinitiated DNA synthesis and the enhanced transport of specific small molecules stimulated by serum in quiescent 3T3 cells. Cytochalasin at concentrations of less than 1 mug/ml inhibits serum-stimulated movement within the monolayer and migration into a wound. Even at ten times this concentration there is little effect on the increase in DNA in the culture, indicating that movement away from neighboring cells is not required for the initiation of DNA synthesis. While DNA synthesis is not inhibited by concentrations of cytochalasin up to 10 mug/ml, the increased thymidine transport which is associated with the onset of the S phase of the cell cycle is inhibited and DNA synthesis cannot be measured by the labelling of nuclei with radioactive thymidine. Cytochalasin has a differential effect on the early transport changes produced by serum addition. Glucose transport is inhibited by low concentrations of the drug (less than 1 mug/ml) while the enhanced uptake of phosphate and uridine is unaffected by a 10-fold increase in concentration. Although the doses of cytochalasin required for 50% inhibition of hexose uptake and of cell movement are the same, no causal relationship between sugar transport and locomotion can be demonstrated. Cytochalasin affects membrane functions in at least two different ways. The drug inhibits the uptake of glucose directly but affects only the S-phase associated increase in thymidine transport.