The host cell chaperone Hsp90 is essential for translocation of the binary Clostridium botulinum C2 toxin into the cytosol

Clostridium botulinum C2 toxin is the prototype of the binary actin-ADP-ribosylating toxins and consists of the binding component C2II and the enzyme component C2I. The activated binding component C2IIa forms heptamers, which bind to carbohydrates on the cell surface and interact with the enzyme component C2I. This toxin complex is taken up by receptor-mediated endocytosis. In acidic endosomes, heptameric C2IIa forms pores and mediates the translocation of C2I into the cytosol. We report that the heat shock protein (Hsp) 90-specific inhibitors, geldanamycin or radicicol, block intoxication of Vero cells, rat astrocytes, and HeLa cells by C2 toxin. ADP-ribosylation of actin in the cytosol of toxin-treated cells revealed that less active C2I was translocated into the cytosol after treatment with Hsp90 inhibitors. Under control conditions, C2I was localized in the cytosol of toxin-treated rat astrocytes, whereas geldanamycin blocked the cytosolic distribution of C2I. At low extracellular pH (pH 4.5), which allows the direct translocation of C2I via C2IIa heptamers across the cell membrane into the cytosol, Hsp90 inhibitors retarded intoxication by C2I. Geldanamycin did not affect toxin binding, endocytosis, and pore formation by C2IIa. The ADP-ribosyltransferase activity of C2I was not affected by Hsp90 inhibitors in vitro. The cytotoxic actions of the actin-ADP-ribosylating Clostridium perfringens iota toxin and the Rho-ADP-ribosylating C2-C3 fusion toxin was similarly blocked by Hsp90 inhibitors. In contrast, radicicol and geldanamycin had no effect on anthrax lethal toxin-induced cytotoxicity of J774-A1 macrophage-like cells or on cytotoxic effects of the glucosylating Clostridium difficile toxin B in Vero cells. The data indicate that Hsp90 is essential for the membrane translocation of ADP-ribosylating toxins delivered by C2II.     

Cell-mediated reduction of the interfragment disulfide in nicked diphtheria toxin. A new system to study toxin entry at low pH

When 125I-labeled nicked diphtheria toxin bound to Vero cells was exposed to pH less than 5.0, a small fraction was reduced to yield A- and B-fragments. The pH required for reduction correlates well with that required to induce intoxication, and the amount of A-fragment released was of the same order as that required to intoxicate the cells. Conditions that protect cells against intoxication, such as acidification of the cytosol, treatment with anion transport inhibitors, or treatment with anti-diphtheria toxin antibodies, prevented the reduction of the interfragment disulfide in cell-bound toxin. In vitro, thioredoxin reduced nicked diphtheria toxin only at pH 5.0 and lower, and the reduction was inhibited by anti-toxin antibodies. This indicates that a conformational change in the toxin, necessary for reduction by the thioredoxin system, is prevented by the antibodies. Reduction by glutathione and cysteine was most efficient at neutral pH and was not inhibited by anti-toxin. The results are consistent with the possibility that cell-mediated reduction of the interfragment disulfide is a measure of the entry of fragment A into the cytosol.     

Control of membrane permeability in animal cells by divalent cations

The permeability of several cell lines, including HeLa, L929, 3T6 and 3T3, to various compounds is affected by the concentration of divalent cations in the culture medium. In the absence of Mg2+ ions but with 4-8 mM CaCl2 in the medium, HeLa and L929 cells become permeabilized, as measured by the entry of the aminoglycoside antibiotic hygromycin B. However, 3T3 and 3T6 cells become much more permeable when calcium and magnesium are both absent from the medium. Addition of Mg2+ above 2 mM abolishes the permeabilization induced by Ca2+. Basic pH favors permeabilization, whereas acidic pH inhibits the entry of hygromycin B. Increased entry of macromolecules, such as the toxin alpha-sarcin, horseradish peroxidase (HRP) and luciferase, is also observed under permeabilization conditions, suggesting that this method could be of general use, since it is not harmful to cells and is fully reversible. Exit of 86Rb+ ions and [3H]uridine-labelled nucleotides was also assayed. We did not observe increased release of these compounds from preloaded cells under various calcium concentrations. Finally, the effects of several inhibitors of endocytosis and other membrane functions on the permeabilization inhibitors of endocytosis and other membrane functions on the permeabilization process were also analysed. The entry of alpha-sarcin was not affected by nifedipine, dibucaine or mepacrine, but was partially inhibited by NH4Cl, amantadine and chloroquine.     

Endocytosis from coated pits of Shiga toxin: a glycolipid-binding protein from Shigella dysenteriae 1

Evidence is presented that endocytosis is involved in the transport to the cytosol of the cytotoxin from Shigella dysenteriae 1, Shiga toxin, which acts by removal of a single adenine residue in 28-S ribosomal RNA. Inhibition of endocytosis by ATP depletion of the cells prevented toxin uptake. Exposure of HeLa S3 and Vero cells to toxin at low extracellular pH, where translocation to the cytosol, but not endocytosis is inhibited, allowed the toxin to accumulate in a compartment where it was protected against antibodies to the toxin. Upon transfer of the cells to normal medium endocytosed toxin entered the cytosol. Electron microscopical studies of cells exposed at 0 degrees C to a toxin-horseradish peroxidase (HRP) conjugate, or to unconjugated toxin followed by horse antitoxin antibodies and then protein G-gold, revealed that the Shiga toxin binding sites were randomly distributed on the cell surface, without any preference to, for example, coated pits. In contrast, when cells were exposed to toxin at 37 degrees C, the binding sites were preferentially localized in coated pits. The Shiga-HRP conjugate was also seen in endosomes, lysosomes, and in the Golgi region. Endocytosis by the coated pit/coated vesicle pathway was selectively inhibited by acidification of the cytosol. Under these conditions, both the uptake of toxin-HRP conjugates and intoxication of the cells were inhibited. Evidence from the literature as well as our own results suggest that Shiga toxin binding sites are glycolipids. Thus, Shiga toxin appears to be the first example of a lipid-binding ligand that is endocytosed from coated pits.     

Acid activation of Helicobacter pylori vacuolating cytotoxin (VacA) results in toxin internalization by eukaryotic cells

Helicobacter pylori VacA is a secreted toxin that induces multiple structural and functional alterations in eukaryotic cells. Exposure of VacA to either acidic or alkaline pH ('activation') results in structural changes in the protein and a marked enhancement of its cell-vacuolating activity. However, the mechanism by which activation leads to increased cytotoxicity is not well understood. In this study, we analysed the binding and internalization of [125I]-VacA by HeLa cells. We detected no difference in the binding of untreated and activated [125I]-VacA to cells. Binding of acid-activated [125I]-VacA to cells at 4 degrees C was not saturable, and was only partially inhibited by excess unlabelled toxin. These results suggest that VacA binds either non-specifically or to an abundant, low-affinity receptor on HeLa cells. To study internalization of VacA, we used a protease protection assay. Analysis by SDS-PAGE and autoradiography indicated that the intact 87 kDa toxin was internalized in a time-dependent process at 37 degrees C but not at 4 degrees C. Furthermore, internalization of the intact toxin was detected only if VacA was acid or alkaline activated before being added to cells. The internalization of activated [125I]-VacA was not substantially inhibited by the presence of excess unlabelled toxin, but was blocked if cells were depleted of cellular ATP by the addition of sodium azide and 2-deoxy-D-glucose. These results indicate that acid or alkaline pH-induced structural changes in VacA are required for VacA entry into cells, and that internalization of the intact 87 kDa toxin is required for VacA cytotoxicity.     

Purification of Bacteroides amylophilus protease

A protease was released by Bacteroides amylophilus cells in late stationary phase, approximately 12 hr after maximum cell density was reached. The protease was concentrated by adsorption on diethylaminoethyl (DEAE)-Sephadex and was purified 532-fold by DEAE-Sephadex chromatography, by G-200-Sephadex gel filtration, and by isoelectric focusing. The purified protease was active between pH 4.5 and 12.0 with optima at pH 6.0 and 11.5. Evidence against there being a single protease was given by the differential inhibition of esterase and protease activities by some inhibitors. There was some evidence that only a single protease was present as the ratio of protease activity at various pH values did not alter significantly during purification or when the purified protease was partially heat-inactivated or treated with two specific trypsin-type protease inhibitors: N-alpha-tosyl-l-lysylchloromethyl ketone or phenylmethane-sulfonyl fluoride. Two forms of the same protease were found by acrylamide gel electrophoresis. Gel filtration confirmed the presence of protease in 30,000 and 60,000 molecular-weight forms. Treatment with 1 mm ethylenediaminetetraacetic acid or with 4 m urea failed to convert the 60,000-molecular-weight to the 30,000-molecular-weight species.     

Formation of anion-selective channels in the cell plasma membrane by the toxin VacA of Helicobacter pylori is required for its biological activity.

The vacuolating toxin VacA, a major determinant of Helicobacter pylori-associated gastric diseases, forms anion-selective channels in artificial planar lipid bilayers. Here we show that VacA increases the anion permeability of the HeLa cell plasma membrane and determines membrane depolarization. Electrophysiological and pharmacological approaches indicated that this effect is due to the formation of low-conductance VacA pores in the cell plasma membrane and not to the opening of Ca(2+)- or volume-activated chloride channels. VacA-dependent increase of current conduction both in artificial planar lipid bilayers and in the cellular system was effectively inhibited by the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), while2-[(2-cyclopentenyl-6,7dichloro-2, 3-dihydro-2-methyl-1-oxo-1H-inden-5-yl)oxy]acetic acid (IAA-94) was less effective. NPPB inhibited and partially reversed the vacuolation of HeLa cells and the increase of ion conductivity of polarized Madine Darby canine kidney cell monolayers induced by VacA, while IAA-94 had a weaker effect. We conclude that pore formation by VacA accounts for plasma membrane permeabilization and is required for both cell vacuolation and increase of trans-epithelial conductivity.     

Toxicity of pseudomonas toxin for mouse LM cell fibroblasts

Pseudomonas toxin inhibited protein synthesis in mouse LM fibroblast monolayers. Incubation of toxin with LM cell monolayers resulted in a depletion of functional elongation factor 2. The initial interaction of pseudomonas toxin with mouse LM cells was rapid; within 2.5 min, toxin was rendered inaccessible to neutralization with specific pseudomonas antitoxin. At 4°C toxin adsorbed to the cell surface, but remained at a site where it could be neutralized with antitoxin. Ammonium chloride (20 mM) rendered LM cells insensitive to the action of toxin. The ammonium salt did not prevent adsorption of toxin to the cell membrane; rather, it appeared to maintain toxin at a site amenable to antitoxin neutralization.     

Clostridium difficile toxin B activates dual caspase-dependent and caspase-independent apoptosis in intoxicated cells

Clostridium difficile toxin B (TcdB) inactivates the small GTPases Rho, Rac and Cdc42 during intoxication of mammalian cells. In the current work, we show that TcdB has the potential to stimulate caspase-dependent and caspase-independent apoptosis. The apoptotic pathways became evident when caspase-3-processed-vimentin was detected in TcdB-treated HeLa cells. Caspase-3 activation was subsequently confirmed in TcdB-intoxicated HeLa cells. Interestingly, caspase inhibitor delayed TcdB-induced cell death, but did not alter the time-course of cytopathic effects. A similar effect was also observed in MCF-7 cells, which are deficient in caspase-3 activity. The time-course to cell death was almost identical between cells treated with TcdB plus caspase inhibitor and cells intoxicated with the TcdB enzymatic domain (TcdB1-556). Unlike TcdB treated cells, intoxication with TcdB1-556 or expression of TcdB1-556 in a transfected cell line, did not stimulate caspase-3 activation yet cells exhibited cytopathic effects and cell death. Although TcdB1-556 treated cells did not demonstrate caspase-3 activation these cells were apoptotic as determined by differential annexin-V/propidium iodide staining and nucleosomal DNA fragmentation. These data indicate TcdB triggers caspase-independent apoptosis as a result of substrate inactivation and may evoke caspase-dependent apoptosis due to a second, yet undefined, activity of TcdB. This is the first example of a bacterial virulence factor with the potential to stimulate multiple apoptotic pathways in host cells.     

Requirement of specific receptors for efficient translocation of diphtheria toxin A fragment across the plasma membrane

The role of specific receptors in the translocation of diphtheria toxin A fragment to the cytosol and for the insertion of the B fragment into the cell membrane was studied. To induce nonspecific binding to cells, toxin was either added at low pH, or biotinylated toxin was added at neutral pH to cells that had been treated with avidin. In both cases large amounts of diphtheria toxin became associated with the cells, but there was no increase in the toxic effect. There was also no increase in the amount of A fragment that was translocated to the cytosol, as estimated from protection against externally added Pronase E. In cells where specific binding was abolished by treatment with 12-O-tetradecanoyl-phorbol 13-acetate, trypsin, or 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, unspecific binding did not induce intoxication or protection against protease. This was also the case in untreated L cells, which showed no specific binding of the toxin. When Vero cells with diphtheria toxin bound to specific receptors were exposed to low pH, the cells were permeabilized to K+, whereas this was not the case when the toxin was bound nonspecifically at low pH or via avidin-biotin. The data indicate that the cell-surface receptor for diphtheria toxin facilitates both insertion of the B fragment into the cell membrane and translocation of the A fragment to the cytosol.     

Zebularine inhibits the growth of HeLa cervical cancer cells via cell cycle arrest and caspase-dependent apoptosis

Zebularine (Zeb) as a DNA methyltrasferase (DNMT) inhibitor has various cellular effects such as cell growth inhibition and apoptosis. In the present study, we evaluated the effects of Zeb on the growth and death of HeLa cervical cancer cells. Zeb inhibited the growth of HeLa cells with an IC(50) of approximately 130?μM at 72?h in a dose-dependent manner. DNA flow cytometric analysis indicated that Zeb induced an S phase arrest of the cell cycle, which was accompanied by the increased levels of cdk2 and cyclin A proteins. This agent also induced apoptosis, which was accompanied by the loss of mitochondrial membrane potential (Ψ(m)), PARP-1 cleavage and the activation of caspase-3, -8 and -9. All of the tested caspase inhibitors significantly rescued some cells from Zeb-induced HeLa cell death. In relation to reactive oxygen species (ROS) and glutathione (GSH) levels, O (2) (?-) level was significantly increased in 100?μM Zeb-treated HeLa cells and caspase inhibitors reduced O (2) (?-) level in these cells. Zeb induced GSH depletion in HeLa cells, which was attenuated by caspase inhibitors. In conclusion, this is the first report that Zeb inhibited the growth of HeLa cells via cell cycle arrest and apoptosis.     

Monoclonal antibody against diphtheria toxin. Effect on toxin binding and entry into cells

A number of monoclonal antibodies against diphtheria toxin were isolated. Some of their properties were determined. Antibody 2 reacts with the region of between 30 and 45 kDa from the NH2 terminus of toxin. Antibody 7 reacts with the COOH-terminal 17-kDa region of toxin. These two antibodies show sharp contrasts in their effects on toxin action in cultured cells. When antibody 2 or 7 and toxin were mixed, incubated at 37 degrees C, and then added to sensitive Vero cells, antibody 7 blocked toxin action, but antibody 2 did not. When antibody 2 or 7 was added to cells to which toxin had been prebound at 4 degrees C, and the cells were then shifted to 37 degrees C, antibody 7 did not block toxin action, but antibody 2 inhibited intoxication. Antibody 7 blocked binding of 125I-toxin to cells and did not block degradation of toxin associated with cells. Antibody 2 did not block binding of 125I-toxin to cells, and was able to bind to cells in the presence of toxin. The results obtained from the effect of antibody 2 on degradation of 125I-toxin associated with cells resemble those seen with amines, which block toxin action but do not inhibit binding of toxin to cells. These facts show that antibody 2 does not block binding of toxin to cell surfaces, but blocks the entry of toxin into the cytosol at a step after binding of toxin to the receptor. Antibodies 14 and 15 react with fragment A of diphtheria toxin, but have no effect on any activity of toxin. The other monoclonal antibodies have effects on toxin binding and entry intermediate between those of 2 and 7.     

Cholesterol-dependent pore formation of Clostridium difficile toxin A

The large clostridial cytotoxins toxin A and toxin B from Clostridium difficile are major virulence factors known to cause antibiotic-associated diarrhea and pseudomembranous colitis. Both toxins mono-glucosylate and thereby inactivate small GTPases of the Rho family. Recently, it was reported that toxin B, but not toxin A, induces pore formation in membranes of target cells under acidic conditions. Here, we reassessed data on pore formation of toxin A in cells derived from human colon carcinoma. Treatment of 86Rb+-loaded cells with native or recombinant toxin A resulted in an increased efflux of radioactive cations induced by an acidic pulse. The efficacy of pore formation was dependent on membrane cholesterol, since cholesterol depletion of membranes with methyl-beta-cyclodextrin inhibited 86Rb+ efflux, and cholesterol repletion reconstituted pore-forming activity of toxin A. Similar results were obtained with toxin B. Consistently, methyl-beta-cyclodextrin treatment delayed intoxication of cells in a concentration-dependent manner. In black lipid membranes, toxin A induced ion-permeable pores only in cholesterol containing bilayers and at low pH. In contrast, release of glycosylphosphatidylinositol-anchored structures by phosphatidylinositol specific phospholipase C treatment did not reduce cell sensitivity toward toxins A and B. These data indicate that in colonic cells toxin A induces pore formation in an acidic environment (e.g. endosomes) similar to that reported for toxin B and suggest that pore formation by clostridial glucosylating toxins depends on the presence of cholesterol.     

Alteration in tRNA Methyltransferase Activity in Mengovirus Infection: Host Range Specificity

The tRNA methyltransferase activity in mengovirus-infected L cells, HeLa cells, and Maden Derby bovine kidney cells has been examined during the course of infection. The first two cell lines yield a productive infection, but have different kinetics of inhibition of host RNA synthesis, whereas the bovine kidney cells are a restrictive host. In infected L cells the enzymes show altered capacity and base specificity throughout the infection. In infected HeLa cells and in infected bovine kidney cells less marked changes were seen. No inhibitors or activators of the enzymes were detected in any of the infected cell lines. Labeling experiments in infected cells indicated that in infected L cells synthesis of RNA was inhibited to a greater degree than was methylation of RNA. The consequence of this would be a hypermethylation of RNA. The methylated derivatives synthesized in infected L cells showed changes in relative proportions. Infection of HeLa cells and bovine kidney cells did not show such marked effects on methylation of RNA.     

Tetanus toxin binds with high affinity to neuroblastoma x glioma hybrid cells NG 108-15 and impairs their stimulated acetylcholine release

Differentiated neuroblastoma x glioma hybrid cells NG 108-15 express on their surface specific binding sites for tetanus toxin. 450 sites/cell with a KD of 2 x 10(-11) M were found under "physiological" conditions of pH and salt concentrations. A Hill coefficient of 1.1 indicated noncooperative binding. Specific binding of 125I-toxin to its sites could be prevented either by preincubation of the toxin with a neutralizing monoclonal antibody or by pretreatment of the cells with neuraminidase (Vibrio cholerae). To quantify the action of tetanus toxin on the stimulated release of 14C activity from differentiated cells preincubated with [14C]choline, a new type of perfusion device was designed which could be filled with cells growing in monolayers on Cytodex-3 microbeads. Tetanus toxin inhibited the stimulated 14C release in a time- and dose-dependent manner. A greater than 50% inhibition was found after 2 h of incubation with 10(-12) M toxin. The inhibitory action of tetanus toxin could be prevented with a monoclonal antibody to the toxin or with neuraminidase treatment of the cells. These results suggest that the neuraminidase-sensitive 2 x 10(-11) KD receptors are the productive receptors for tetanus intoxication in differentiated NG 108-15 cells. The possible chemical composition of these receptors is discussed. Differentiated NG 108-15 cells provide a useful model in which picomolar tetanus concentrations produce both measurable saturable binding and inhibition of potassium-evoked, acetylcholine release under physiological conditions of pH and salt concentrations.     

Dual role of GTP-binding proteins in the control of endothelial prostacyclin

Pretreatment of bovine aortic endothelial cells with pertussis toxin inhibited partially the accumulation of inositol phosphates in response to ATP, whereas cholera toxin had no effect. Both pertussis and cholera toxins enhanced the stimulatory effect of ATP on prostacyclin release from the same cells. This action of cholera toxin was mimicked neither by an increase of cyclic AMP nor by the dissociated subunits of the toxin. Cholera and pertussis toxins, as well as aluminum fluoride, also potentiated the release of prostacyclin induced by ionophore A23187. These results suggest that a pertussis toxin-sensitive GTP-binding protein is involved in the coupling between P2-purinergic receptors and phospholipase C. In addition, another GTP-binding protein would play a crucial role at a further step in the control of PGI2 biosynthesis.     

Calcium and calmodulin in cellular intoxication with Clostridium difficile toxin B

In cultured human lung fibroblasts treated with Clostridium difficile toxin B, the development of the cytopathogenic effect was inhibited by the proton ionophore monensin but was not affected by some other ionophores. The calcium channel blockers verapamil and LaCl3 protected the cells against intoxication, as did the calmodulin antagonists trifluoperazine, amitriptyline, R 24571, and dansylcadaverine. Since these agents could not prevent intoxication when added after the toxin internalization was completed, we suggest that calmodulin and uptake of extracellular calcium are needed for the internalization but not for the cytosolic action of the toxin.     

The enhancement of propyl gallate-induced HeLa cell death by MAPK inhibitors is accompanied by increasing ROS levels

Propyl gallate (PG) as a synthetic antioxidant exerts a variety of effects on tissue and cell functions. Here, we investigated the effects of MAPK (MEK, JNK and p38) inhibitors on PG-treated HeLa cells in relation to cell death, ROS and GSH levels. PG induced cell growth inhibition and apoptosis in HeLa cells, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m). ROS levels were increased or decreased in PG-treated HeLa cells depending on the incubation times. PG also increased GSH depleted cell numbers in HeLa cells. All the MAPK inhibitors slightly enhanced cell growth inhibition, death and MMP (ΔΨ_m) loss, and increased ROS levels in PG-treated HeLa cells. However, MAPK inhibitors did not significantly affect GSH depletion in PG-treated cells. In conclusion, the enhanced effect of MAPK inhibitors on PG-induced HeLa cell death was accompanied by increasing ROS levels but the effect was not related to changes of GSH level.     

Triflavin, an Arg-Gly-Asp-containing peptide, inhibits human cervical carcinoma (HeLa) cell-substratum adhesion through an RGD-dependent mechanism

Triflavin, a 7.5-kDa cysteine-rich polypeptide purified from Trimeresurus flavoviridis snake venom, belongs to a family of RGD-containing peptides, termed disintegrins, that have been isolated from the venoms of various vipers and shown to be potent inhibitors of platelet aggregation. The interaction of tumor cells with extracellular matrices such as fibronectin, vitronectin, and collagen has been shown to be mediated through a family of cell surface receptors that specifically recognize an arginine-glycine-aspartic acid (RGD) sequence within each adhesive protein. In this study, we show that triflavin dose-dependently inhibited adhesion of human cervical carcinoma (HeLa) cells to extracellular matrices (ECMs; i.e., fibronectin, fibrinogen, and vitronectin). On the other hand, triflavin exerted a limited inhibitory effect on cell adhesion to laminin and collagen (type I and IV). On a molar basis, triflavin is approximately 800 times more potent than Gly-Arg-Gly-Asp-Ser (GRGDS) at inhibiting cell adhesion. When immobilized on plate, triflavin significantly promoted HeLa cell adhesion, and this attachment was inhibited by GRGDS. Furthermore, FITC-conjugated triflavin bound to cells in a saturable manner and its binding was inhibited by GRGDS. In addition, triflavin did not affect [³H]thymidine uptake of HeLa cells during a 3-day incubation. These results suggest that triflavin probably binds to integrin receptors expressed on HeLa cell surface via its RGD sequence within its molecule, thereby inhibiting the adhesion of extracellular matrices to HeLa cells.     

Correlation between cell enlargement and nucleic acid and protein content of HeLa cells in unbalanced growth produced by inhibitors of DNA synthesis

HeLa cells in monolayer cultures were treated with the following inhibitors of DNA synthesis: mitomycin C, nitrogen mustard, fluorodeoxyuridine, hydroxyurea, arabinofuranosylcytosine and high concentrations of thymidine. The concentration of each inhibitor used was, in most cases, just sufficient to arrest cell multiplication and all produced unbalanced growth in the sense that the synthesis of RNA and protein were only partially inhibited while DNA synthesis stopped. This resulted in approximately 100% increases in RNA and protein content per cell in 48 hours and, since cell volume also increased by 100% during this time, the concentration of RNA and protein per unit cell volume remained constant. It was concluded that cell protein content may be used as an accurate index of variation in cell size in HeLa cells treated with inhibitors of DNA synthesis.