Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological or Breast Cancers

Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not initiated by brain mononuclear cells. Purkinje cell death was not simply due to intraneuronal antibody accumulation.     

Copper biosorption by Pseudomonas cepacia and other strains

Biosorption of copper by Pseudomonas cepacia was found to be dependent on added copper concentration. Copper uptake by the cells was rapid over the range of copper concentrations tested and complete within the first 10 min of incubation time. The effect of pH on copper uptake by P. cepacia was determined using overlapping buffers over the pH range 3–8, and copper biosorption from a 10 mM copper solution was greatest at pH 7. Copper uptake (measured by analysis of cell digests) was unaffected by cyanide and azide (up to 30 mM) and by incubation of cells with a 10 mM copper solution at 4 °C. Evidence from these results suggested that copper uptake by P. cepacia cells involves surface binding and not intracellular accumulation by active transport. Biosorption of copper by various Pseudomonas isolates from metal-contaminated environments agreed well with copper biosorption by Pseudomonas strains from the National Collection of Type Cultures (NCTC).     

Immobilization Results in Sustained Calcium Transport in Nostoc calcicola Bréb

The uptake pattern of Ca²⁺ by the cyanobacterium Nostoc calcicola Bréb in its freely suspended and immobilized form is comprised of two distinct phages; (a) rapid uptake for 1^st 10 min followed by (b) slower transport at least up to 60 min. Entrapment of cyanobacterial cells in polyvinyl foam always maintained a higher Ca²⁺ profile over freely suspended cells. Also, the intracellular Ca²⁺ concentration was three times more in the former under similar experimental conditions. Whereas, illumination supported maximum Ca²⁺ transport in all the sets, darkness resulted in drastic reduction (90%) of Ca²⁺ uptake in freely suspended cells and least (15%) in polyvinyl entrapped cyanobacterial cells. Exogenously added ATP (10 μM) on the other hand, enhanced Ca²⁺ uptake in dark incubated freely suspended cells; ATP at the same concentration failed to bring out any significant enhancement in cation uptake in immobilized cells facing dark exposure. It was observed that these cells were still able to sustain sufficient ATP preserves to drive active transport of Ca²⁺ even in the dark. Furthermore, the immobilized cells exhibited remarkable Ca²⁺ transport rate even at the age of 20 and 50 days at which its free living counterpart took up insignificant Ca²⁺. These findings suggest the improved metabolic efficiency of polyvinyl foam entrapped cells over freely suspended cells in terms of Ca²⁺ accumulation and its possible use as a bioreactor for metal accumulation/removal in repetitive cycles without any measurable loss in cell biomass. Received: 21 May 2001 / Accepted: 27 June 2001     

Biodegradation of pyridine by freely suspended and immobilized Pimelobacter sp.

Freely suspended and Ca-alginate-immobilized cells of Pimelobacter sp. were used for degradation of pyridine. When the pyridine concentration was up to 2 g l^–1, freely suspended cells completely degraded pyridine regardless of the initial cell concentrations used. However, when the pyridine concentration increased to 4 g l^–1, the initial cell concentration in freely suspended cell culture should be higher than 1.5 g dry cell weight l^–1 for complete degradation of pyridine. In addition, a freely suspended cell culture with a high initial cell concentration resulted in a high volumetric pyridine-degradation rate, suggesting the potential use of immobilized cells for pyridine-degradation. When the immobilized cells were used for pyridine-degradation, neither specific pyridine-degradation rate nor tolerance against pyridine was improved. However, a high volumetric pyridine-degradation rate in the range 0.082–0.129 g l^–1 hr^–1 could be achieved by the immobilized cells because of the high cell concentration. Furthermore, when the immobilized cells were reused in degrading pyridine at a concentration of 2–4 g l^–1 they did not lose their pyridine-degrading activity for 2 weeks. Taken together, the data obtained here showed the feasibility of using immobilized cells for pyridine-degradation.     

Methods to measure the intracellular concentration of unlabeled compounds within cultured cells using liquid chromatography/tandem mass spectrometry

A high-throughput and sensitive liquid chromatography/tandem mass spectrometry assay was established to detect total unlabeled hepatitis C virus inhibitor concentrations in replicon cells. The intracellular concentrations determined by this assay correlated well with concentrations obtained using radiolabeled compound. Some compounds accumulated inside the cells, with concentrations up to 300-fold higher than the input concentration. Confocal microscopic evaluation of two fluorescent-tagged inhibitors confirmed high accumulation inside the cells, sequestered inside vesicles within the cytoplasm. Incubation of cells with compound at 4 °C revealed that nonspecific binding to the outside of the cell membrane and to the cell culture plate occurred for some compounds. Therefore, the total concentration of compound extracted at 37 °C was reduced by the amount that was nonspecifically bound at 4 °C to yield the amount of compound inside the cells. A modification of the protocol was used for compounds with low intracellular concentrations in which cells were harvested with trypsin-EDTA prior to extraction. This eliminated the nonspecific binding to the cell culture plate and decreased the overall background of the assay. This assay was used to understand differences in cellular potency between compounds and the effects of serum proteins on the metabolic stability of compounds during incubation with cells.     

Effect of cations,including heavy metals,on cadmium uptake by Lemna polyrhiza L.

Cations, including calcium, magnesium, potassium, sodium, copper, iron, nickel and zinc, inhibited (up to 40%) extracellular binding and intracellular uptake of cadmium by Lemna polyrhiza in solution culture. Test plants showed a high capacity of extracellular cadmium binding which was competitively inhibited by copper, nickel and zinc; however, calcium, magnesium and potassium caused non-competitive inhibition. Iron and sodium increased K _m and decreased V _max, thereby causing mixed inhibition of extracellular binding. Intracellular cadmium uptake displayed Michaelis-Menten kinetics. It was competitively inhibited by calcium, magnesium, iron, nickel and zinc. Monovalent cations (sodium and potassium) caused non-competitive and copper caused mixed inhibition of intracellular cadmium uptake. Thus, high levels of cations and metals in the external environment should be expected to lower the cadmium accumulation efficiency of L. polyrhiza.     

The biochemical and ultrastructural effects of tunicamycin and D-glucosamine in L1210 leukemic cells

Tunicamycin was found to specifically inhibit the incorporation of a number of sugars into L1210 leukemia cell glycoproteins. This inhibition of glyco-protein biosynthesis led to a cessation of cell growth which was reversible in a dose-dependent and time-dependent manner. After removal of the antibiotic from L1210 cell cultures resumption of sugar incorporation preceded that of thymidine incorporation and the recovery of cell growth. The treatment of cells with tunicamycin resulted in a significant increase in the intracellular pool of UDP-N-acetylglucosamine which occurred concurrently with alterations in cell ultrastructure including distentions of the endoplasmic reticulum and nuclear membranes. Similar ultrastructural changes and increases in the intracellular pools of UDP-sugars were observed in L1210 cells exposed to 5 mM D-glucosamine, which suggested that the antiproliferative effects of tunicamycin may be related to the accumulation in the endoplasmic reticulum of one or more nucleotide sugar precursors of asparagine-linked glycoprotein biosynthesis. However, the biological effects of tunicamycin could be distinguished from those caused by D-glucosamine. Exposure of L1210 cells to tunicamycin resulted in specific alterations in the biochemical composition of the plasma membrane and in the inhibition of cellular agglutination by wheat germ agglutinin which were not apparent following exposure to equitoxic concentrations of the aminosugar. These studies, together with those which demonstrated that recovery of the cellular capacity to synthesize glycoproteins was obligatory for the recovery of cellular proliferation in tunicamycin-treated cells, suggested that inhibition of the synthesis of glycoproteins was the major factor limiting L1210 leukemic cell proliferation.     

Effects of dichloroacetate and glyoxylate on low density lipoprotein uptake and on growth of cultured fibroblasts

The effects of dichloroacetate, a known hypocholesterolemic agent, were studied in cultured growing and confluent human fibroblast cells. Microscopic examination showed no visible adverse effects of dichloroacetate on confluent cells during exposure to concentrations as high as 5 mM for 96 hr. Higher concentrations resulted in cell death after varying periods of incubation. There were no viable cells after 24 hr of exposure to 100 mM dichloroacetate. In contrast, much lower concentrations proved lethal to growing cells; cell growth, as determined by cell numbers at specified times after splitting, was suppressed by 1 mM dichloroacetate and 5 mM concentrations resulted in cell death. Similar effects were noted with glyoxylate. The hypocholesterolemic effect of dichloroacetate is probably not due to any effect on the low density lipoprotein pathway, since concentrations of up to 1 mM dichloroacetate did not affect the cellular binding and uptake of 125I-labeled low density lipoprotein. It is concluded that growing and rapidly metabolizing cells are much more sensitive to the toxic effects of dichloroacetate and glyoxylate than confluent cells.     

Vanadate down-regulates cell surface insulin and growth hormone receptors and inhibits insulin receptor degradation in cultured human lymphocytes

Insulin is able to down-regulate its specific cell surface receptor in cultured human lymphocytes. The effect of vanadate, a known insulinomimetic agent, was examined to determine whether it could mimic insulin to down-regulate the insulin receptor. Exposure of cultured human lymphocytes (IM-9) to vanadate (0-200 microM) resulted in a time- and dose-dependent decrease in cell surface insulin receptors to 60% of control, while insulin (100 nM) down-regulated to 40%. The vanadate effect, in contrast to the rapid effect of insulin, was slow to develop (4-6 h). Surface receptor recovery after 18 h exposure was rapid after vanadate removal (20 min), but it required hours after insulin suggesting the presence of an intracellular (cryptic) pool of receptors after vanadate treatment. Insulin binding to Triton X-100-solubilized whole cells after 18 h treatment revealed that total cell receptors had decreased to 50% of control after insulin but increased to 120 and 189% of control after 100 and 200 microM vanadate, respectively. Furthermore, vanadate inhibited the insulin-mediated loss of total cell receptors from 50 to 28%. Removal of cell surface receptors by trypsin before cell solubilization revealed that 100 microM vanadate increased insulin binding to 321% of control indicating an accumulation of intracellular receptors. Labeling of cell surface proteins with Na125I and lactoperoxidase followed by immunoprecipitation of solubilized receptors with anti-receptor antibody after incubation for various times up to 20 h and quantitation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, while insulin shortened t1/2 from 7.3 to 5.3 h, vanadate prolonged receptor t1/2 to 14 h. No effect of vanadate was detected on insulin receptor tyrosine kinase activity with up to 4 h incubation at the vanadate concentrations used in this study. Furthermore, human growth hormone surface receptors were similarly down-regulated by vanadate. We conclude that 1) vanadate has an apparent insulin-like effect to down-regulate cell surface insulin receptors in cultured human lymphocytes; 2) in contrast to insulin-induced down-regulation which is associated with receptor degradation vanadate causes an accumulation of intracellular (cryptic) receptors and inhibits insulin receptor degradation; and 3) these effects of vanadate may be exerted on other cell surface receptors.     

A reliable method for detecting the intracellular accumulation of fermentation products: Application to intracellular ethanol analysis

Summary Intracellular ethanol concentrations inSaccharomyces cerevisiae were measured using a high cell density fermentation technique which permits samples to be fixed for analysis in less than two seconds. No accumulation of intracellular ethanol was detected, regardless of the stage of fermentation. The technique eliminates artifacts associated with concentrating the cell sample and provides a reliable means for detecting whether other fermentation products accumulate intracellularly.     

High molecular weight kininogen utilizes heparan sulfate proteoglycans for accumulation on endothelial cells

Kininogens, the high molecular weight precursor of vasoactive kinins, bind to a wide variety of cells in a specific, reversible, and saturable manner. The cell docking sites have been mapped to domains D3 and D5(H) of kininogens; however, the corresponding cellular acceptor sites are not fully established. To characterize the major cell binding sites for kininogens exposed by the endothelial cell line EA.hy926, we digested intact cells with trypsin and other proteases and found a time- and concentration-dependent loss of (125)I-labeled high molecular weight kininogen (H-kininogen) binding capacity (up to 82%), indicating that proteins are crucially involved in kininogen cell attachment. Cell surface digestion with heparinases similarly reduced kininogen binding capacity (up to 78%), and the combined action of heparinases and trypsin almost eliminated kininogen binding (up to 85%), suggesting that proteoglycans of the heparan sulfate type are intimately involved. Consistently, inhibitors such as p-nitrophenyl-beta-d-xylopyranoside and chlorate interfering with heparan sulfate proteoglycan biosynthesis reduced the total number of kininogen binding sites in a time- and concentration-dependent manner (up to 67%). In vitro binding studies demonstrated that biotinylated H-kininogen binds to heparan sulfate glycosaminoglycans via domains D3 and D5(H) and that the presence of Zn(2+) promotes this association. Cloning and over-expression of the major endothelial heparan sulfate-type proteoglycans syndecan-1, syndecan-2, syndecan-4, and glypican in HEK293t cells significantly increased total heparan sulfate at the cell surface and thus the number of kininogen binding sites (up to 3. 3-fold). This gain in kininogen binding capacity was completely abolished by treating transfected cells with heparinases. We conclude that heparan sulfate proteoglycans on the surface of endothelial cells provide a platform for the local accumulation of kininogens on the vascular lining. This accumulation may allow the circumscribed release of short-lived kinins from their precursor molecules in close proximity to their sites of action.     

High-Affinity Accumulation of a Maytansinoid in Cells via Weak Tubulin Interaction

The microtubule-targeting maytansinoids accumulate in cells and induce mitotic arrest at 250- to 1000-fold lower concentrations than those required for their association with tubulin or microtubules. To identify the mechanisms of this intracellular accumulation and exceptional cytotoxicity of maytansinoids we studied interaction of a highly cytotoxic maytansinoid, S-methyl DM1 and several other maytansinoids with cells. S-methyl DM1 accumulated inside the cells with a markedly higher apparent affinity than to tubulin or microtubules. The apparent affinities of maytansinoids correlated with their cytotoxicities. The number of intracellular binding sites for S-methyl DM1 in MCF7 cells was comparable to the number of tubulin molecules per cell (~ 4–6 × 10⁷ copies). Efflux of ³ [H]-S-methyl DM1 from cells was enhanced in the presence of an excess of non-labeled S-methyl DM1, indicating that re-binding of ³ [H]-S-methyl DM1 to intracellular binding sites contributed to its intracellular retention. Liposomes loaded with non-polymerized tubulin recapitulated the apparent high-affinity association of S-methyl DM1 to cells. We propose a model for the intracellular accumulation of maytansinoids in which molecules of the compounds diffuse into a cell and associate with tubulin. Affinities of maytansinoids for individual tubulin molecules are weak, but the high intracellular concentration of tubulin favors, after dissociation of a compound-tubulin complex, their re-binding to a tubulin molecule, or to a tip of a microtubule in the same cell, over their efflux. As a result, a significant fraction of microtubule tips is occupied with a maytansinoid when added to cells at sub-nanomolar concentrations, inducing mitotic arrest and cell death.     

Influence of External pH and Fermentation Products on Clostridium acetobutylicum Intracellular pH and Cellular Distribution of Fermentation Products

Clostridium acetobutylicum ATCC 824 cells harvested from a phosphate-limited chemostat culture maintained at pH 4.5 had intracellular concentrations of acetate, butyrate, and butanol which were 13-, 7-, and 1.3-fold higher, respectively, than the corresponding extracellular concentrations. Cells from a culture grown at pH 6.5 had intracellular concentrations of acetate and butyrate which were only 2.2-fold higher than the respective external concentrations. The highest intracellular concentrations of these acids were attained at ca. pH 5.5. When cells were suspended in anaerobic citrate-phosphate buffer at pH 4.5, exogenous acetate and butyrate caused a concentration-dependent decrease in the intracellular pH, while butanol had relatively little effect until the external concentration reached 150 mM. Acetone had no effect at concentrations up to 200 mM. These data demonstrate that acetate and butyrate are concentrated within the cell under acidic conditions and thus tend to lower the intracellular pH. The high intracellular butyrate concentration presumably leads to induction of solvent production, thereby circumventing a decrease in the intracellular pH great enough to be deleterious to the cell.     

Cadmium accumulation and interactions with zinc, copper, and manganese, analysed by ICP-MS in a long-term Caco-2 TC7 cell model

The influence of long-term exposure to cadmium (Cd) on essential minerals was investigated using a Caco-2 TC7 cells and a multi-analytical tool: microwave digestion and inductively coupled plasma mass spectrometry. Intracellular levels, effects on cadmium accumulation, distribution, and reference concentration ranges of the following elements were determined: Na, Mg, Ca, Cr, Fe, Mn, Co, Ni, Cu, Zn, Mo, and Cd. Results showed that Caco-2 TC7 cells incubated long-term with cadmium concentrations ranging from 0 to 10 μmol Cd/l for 5 weeks exhibited a significant increase in cadmium accumulation. Furthermore, this accumulation was more marked in cells exposed long-term to cadmium compared with controls, and that this exposure resulted in a significant accumulation of copper and zinc but not of the other elements measured. Interactions of Cd with three elements: zinc, copper, and manganese were particularly studied. Exposed to 30 μmol/l of the element, manganese showed the highest inhibition and copper the lowest on cadmium intracellular accumulation but Zn, Cu, and Mn behave differently in terms of their mutual competition with Cd. Indeed, increasing cadmium in the culture medium resulted in a gradual and significant increase in the accumulation of zinc. There was a significant decrease in manganese from 5 μmol Cd/l exposure, and no variation was observed with copper.     

Metabolic patterns and insulin responsiveness of enlarging fat cells

The rate and pattern of glucose metabolism, basal lipolysis, and intracellular concentration of free fatty acids were determined in isolated epididymal fat cell preparations (mean volume 30-800 pl) from rats on the basis of fat cell number and in relation to the cell volume. The effects of increasing glucose concentrations in the medium and of insulin on the cellular metabolic activities were compared. Expanding fat cell volume correlated positively and significantly (P < 0.001) with the synthesis of glyceride glycerol from glucose (correlation coefficient, r = 0.919), with rates of basal lipolysis (r = 0.663), and with intracellular free fatty acid accumulation (r = 0.796); it correlated negatively and significantly with glucose conversion to glyceride fatty acids (r = -0.814, P < 0.01). The differences in patterns of glucose metabolism and basal lipolysis between small (<100 pl) and large (>400 pl) fat cells were not modified by insulin or by increments in glucose concentration. The results indicate that the reduced capacity of the large fat cells to respond to insulin cannot be attributed solely to a limited capacity of the cells to take up and metabolize increasing amounts of glucose. The acquired unresponsiveness of the large cells to insulin may result from an alteration in the mechanism of action of insulin and may be related to an intracellular metabolic derangement with increased basal lipolysis, free fatty acid accumulation, and accelerated glyceride synthesis resulting from the accumulation of triglyceride.     

A defect in carbon catabolite repression associated with uncontrollable and excessive maltose uptake

Summary The previously isolated recessive mutant allele hex2-3 of Saccharomyces cerevisiae caused a defect in carbon catabolite repression of maltase, invertase, malate dehydrogenase, and respiration but at the same time led to an extreme sensitivity to maltose (Zimmermann and Scheel, 1977; Entian and Zimmermann, 1980). Addition of maltose to a growing culture of a hex2-3 mutant resulted within 60 to 90 min in an inhibition of growth, glycolysis, and de novo protein synthesis. This was not accompanied by any abnormal levels of glycolysis metabolites or glycolytic enzyme activities. However, inhibitory effects coincided with a dramatic increase in intracellular glucose up to 150 mM relative to cell water as opposed to 2.5 mM in wild-type cells. This abnormal behavior is interpreted as a result of an uncontrolled maltose uptake in hex2 mutants, which in combination with increasing maltase activity results in an accumulation of intracellular glucose. Obviously the amount of available glucose surpassed glycolytic capacity in hex2 mutants.Properties of mutant alleles hex2 and hex1 (see Entian and Zimmermann, 1980) clearly show, that specific gene functions are involved in adapting the rate of sugar uptake into the cell to the actual glycolytic capacity.     

Influence of phenol on biodegradation of p-nitrophenol by freely suspended and immobilized Nocardioides sp. NSP41

The effect of the presence of an alternate toxiccompound (phenol) on the p-nitrophenol(PNP)-degrading activity of freely suspended andcalcium alginate immobilized Nocardioides sp.NSP41 was investigated. In the single substrateexperiments, when the concentration of phenol and PNPwas increased to 1400 mg l^-1 and 400 mg l^-1,respectively, the initial cell concentrations in thefreely suspended cell culture should be higher than1.5 g dry cell weight l^-1 for completedegradation. In the simultaneous degradationexperiment, when the initial concentration of phenolwas increased from 100 to 400 mg l^-1, thespecific PNP degradation rate at the concentration of200 mg l^-1 was decreased from 0.028 to 0.021h^-1. A freely suspended cell culture with a highinitial cell concentration resulted in a highvolumetric degradation rate, suggesting the potentialuse of immobilized cells for simultaneous degradation.In the immobilized cell cultures, althoughsimultaneous degradation of PNP and phenol wasmaintained, the specific PNP and phenol degradationrate decreased. However, a high volumetric PNP andphenol degradation rate could be achieved byimmobilization because of the high cell concentration.Furthermore, when the immobilized cells were reused inthe simultaneous degradation of PNP and phenol, theydid not lose their PNP- and phenol-degrading activityfor 12 times in semi-continuous cultures. Takentogether, the use of immobilized Nocardioidessp. NSP41 for the simultaneous degradation of PNP andphenol at high concentrations is quite feasiblebecause of the high volumetric PNP and phenoldegradation rate and the reusability of immobilizedcells.     

The influence of methotrexate pretreatment on 5-fluorouracil metabolism in L1210 cells

Pretreatment of L1210 cells with methotrexate in concentrations which produced free intracellular methotrexate and near maximal inhibition of dihydrofolate reductase resulted in an enhancement of intracellular 5-fluorouracil (FUra) accumulation. This enhancement of FUra accumulation was maximum (5-fold increase) after a 6-h exposure to 100 microM methotrexate. The nucleotide derivatives of FUra, including a 5-fluoro-2'-deoxyuridylate, and 5-fluorouridine-5'-triphosphate were also increased nearly 5-fold following methotrexate treatment. In cells pretreated with methotrexate, there was an increase in intracellular 5-phosphoribosyl-1-pyrophosphate pools which ranged from 2 to 8 times control values following concentrations of methotrexate between 0.1 microM and 10 microM. Both the increase in 5-phosphoribosyl-1-pyrophosphate and FUra accumulation could be prevented by the addition of Leucovorin (N5-formyltetrahydrofolate) at concentrations which rescued cells from the inhibitory effects of methotrexate. Pretreatment with 6-methylmercaptopurine riboside, which inhibits amidophosphoribosyltransferase, the first committed step in de novo purine synthesis, also resulted in a similar elevation in 5-phosphoribosyl-1-pyrophosphate pools and enhancement of FUra accumulation. If the 5-phosphoribosyl-1-pyrophosphate pools were reduced following methotrexate pretreatment by the addition to the cultures of hypoxanthine, which utilizes 5-phosphoribosyl-1-pyrophosphate for the conversion to IMP, the intracellular accumulation of FUra was not enhanced. Also, if the inhibitor of 5-phosphoribosyl-1-pyrophosphate synthetase, 7-deazaadenosine, was given to cultures with methotrexate, there was no increase in 5-phosphoribosyl-1-pyrophosphate pools, nor enhancement of FUra accumulation. In addition, when 5-fluoro-2'-deoxyuridine was added with the methotrexate to cell cultures, there was no increase in 5-phosphoribosyl-1-pyrophosphate pools, nor enhancement of intracellular FUra accumulation. These results indicate that the ability of methotrexate to enhance FUra accumulation was probably the consequence of the antipurine effect of methotrexate which resulted in a reduction of the complex feedback inhibition on 5-phosphoribosyl-1-pyrophosphate synthesis and utilization. The resultant increased 5-phosphoribosyl-1-pyrophosphate pools were then capable of being utilized for the conversion of FUra to 5-fluorouridylate, the possible rate-limiting step in FUra intracellular metabolism and the major determinant of the rate of intracellular FUra accumulation. When methotrexate preceded FUra, there was synergistic cell killing as determined by soft agar cloning. The exact mechanism of this sequential synergistic antitumor activity may be the result of the enhanced incorporation of FUra into RNA, since the increased 5-fluoro-2'-deoxyuridylate which is formed is unlikely to increase substantially the inhibition of dTMP synthesis induced by methotrexate pretreatment.