Isolation and characterization by electrospray-ionization mass spectrometry and high-performance anion-exchange chromatography of oligosaccharides derived from hyaluronic acid by hyaluronate lyase digestion: observation of some heretofore unobserved oligosaccharides that contain an odd number of units.

Hyaluronic acid was degraded with hyaluronate lyase (E.C. 4.2.2.1, from Streptomyces hyalurolyticus), and the resulting oligosaccharides up to dp 16 were characterized by electrospray-ionization mass spectrometry (ESIMS) and high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD). In accordance with the known regiospecificity of the enzyme, the products included even-numbered oligosaccharides of structure beta-D-4en-thrHexpA-(1-->3)-[beta-D-GlcpNAc-(1-->4)-beta-D- GlcpA]n-(1-->3)-D-GlcpNAc. Minor amounts of novel and unexpected odd-numbered oligomers, having the structure beta-D-4en-thrHexpA-(1-->3)-[beta-D-GlcpNAc-(1-->4)-D-Glc pA]n, were also isolated and characterized. This study, in addition to others beginning to appear in the literature, demonstrates the usefulness of ESIMS and HPAEC-PAD in the analysis and characterization of anionic glycosaminoglycan-type oligosaccharides.

Tiazofurin enhances the anabolism and toxicity of 5-fluorouracil.

Tiazofurin, a clinically active anticancer agent, is undergoing additional clinical testing in combination with other agents. We found that tiazofurin is an effective biochemical modulator of 5-fluorouracil anabolism. Pretreatment of cultured L1210 cells with tiazofurin at concentrations of 1-100 microM results in an increase in the rate of conversion of 5-fluorouracil to phosphorylated metabolites. Concentrations of tiazofurin effective in increasing 5-fluorouracil anabolism cause a corresponding increase in the 5-phosphoribosyl-1-pyrophosphate pool. Studies in mice show that tiazofurin increases the lethal toxicity of 5-fluorouracil and increases the antitumor effectiveness of low doses of 5-fluorouracil: however, the combination is not more effective than an optimal dose of 5-fluorouracil given alone. These results indicate that caution should be exercised in the concurrent use of tiazofurin with other drugs, particularly 5-fluorouracil, that require 5-phosphoribosyl-1-pyrophosphate for activation or that are affected by a decrease in pyrimidine nucleotide synthesis.

Synthesis and release of hyaluronic acid by Swiss 3T3 fibroblasts.

Hyaluronic acid (HA) and its synthesis were studied in intact Swiss 3T3 mouse fibroblasts and isolated membranes. HA chains in culture medium, attached to cells and in isolated membranes, were determined to possess average M(r) values of 5.2 x 10(6), 1.8 x 10(6) and 0.14 x 10(6) respectively. Log cells were determined to possess 680,000 HA molecules/cell, and to release 120,000 HA chains/h. The time required for intact cells to synthesize and release a complete HA chain was approximately 4 h, with elongation proceeding at a rate of 57 dimers/min. The amount of cell-associated HA of various cell populations correlated strongly with their rate of HA release into culture media and with the HA synthetase activity determined for their membranes. Prevention of protein synthesis with cycloheximide decreased the rate of HA synthesis of log cells and HA synthetase activity of isolated membranes by 50% within 2-3 h. Because of the similarity between the biological lifetime of HA synthetase and the time required to synthesize a HA chain, we propose a model where each synthetase makes only one HA chain; after synthesis of a complete HA chain, HA synthetase activity is terminated as its HA chain is released from the cell.

A filter paper assay for hyaluronic acid synthetase: application to the enzyme from Swiss 3T3 fibroblasts.

An improved assay for hyaluronic acid (HA) synthetase is described that is suitable for rapid processing of large numbers of samples. High background levels of unincorporated radioactivity are removed by passage of the reaction through a Sephadex G-50 spin column. The labeled HA product is then precipitated onto glass fiber filters with cetylpyridinium chloride. Apparent Km values for HA synthetase from Swiss 3T3 fibroblasts are 10.8 and 58.4 microM for UDP-glucuronic acid and UDP-N-acetylglucosamine, respectively. HA synthetase activity of quiescent cells is 4.5% of that found in actively growing cells and is stimulated in response to 10% calf serum. There is a greater than 10-fold increase in HA synthetase activity when cells are harvested with hyaluronidase as compared with trypsin.

Cyclopentenyl uracil: an effective inhibitor of uridine salvage in vivo.

Cyclopentenyl uracil, a non-cytotoxic inhibitor of uridine kinase, was found to effectively block the salvage of circulating uridine by host and tumor tissues in the intact mouse. Dose-response characteristics of the inhibition were determined. Large doses (1 g/kg) of cyclopentenyl uracil were required, and the effect of a single dose fell rapidly over a 24-hr period. A sustained inhibition of uridine salvage of > 64-79% could be maintained by multiple doses of 1 g/kg given on an every 8-hr schedule. Mice given cyclopentenyl uracil (1 g/kg) every 8 hr for 5 days continued to gain weight and showed no signs of toxicity; however, the combination of cyclopentenyl uracil with a non-toxic dose of N-(phosphonacetyl)-L-aspartic acid (PALA; 200 mg/kg daily for 5 days) was lethal to mice, indicating that circulating uridine modifies the toxicity of agents that act on enzymes of the de novo pyrimidine pathway. Although the duration of action and potency of cyclopentenyl uracil are not ideal, this is the first demonstration of an effective inhibition of uridine salvage in the intact mouse with a non-cytotoxic agent. This makes possible the evaluation of concurrent inhibition of de novo and salvage routes to pyrimidine nucleotides as an approach to chemotherapy.

Non-steroidal anti-inflammatory drugs and tumor progression: inhibition of fibroblast hyaluronic acid production by indomethacin and mefenamic acid.

The antitumor effects of non-steroidal anti-inflammatory drugs (NSAIDs) have been documented in a variety of both clinical and experimental settings, although the mechanisms responsible remain unclear. In the present study, we show that the NSAIDs indomethacin and mefenamic acid inhibit the calf serum-stimulated production of hyaluronic acid (HA) in murine Swiss 3T3 fibroblasts, at concentrations where DNA synthesis is unaffected. HA is an extracellular matrix glycosaminoglycan associated with cell migration and tumor invasion. Our data suggest that one mechanism whereby NSAIDs inhibit tumor progression may be to inhibit the synthesis of HA by host fibroblasts, and that the eicosenoid pathway may represent an important control point in the growth-factor-mediated production of HA in fibroblasts. Thus the use of an agent which inhibits HA synthesis may be a novel approach to alter the invasive and metastatic properties of tumor cells in a non-cytotoxic fashion.

Vanadate inhibition of hyaluronic acid synthesis in Swiss 3T3 fibroblasts.

Vanadate (50 microM) increases the incorporation of 3H from D-[1,6-3H]glucosamine into hyaluronic acid (HA) in serum stimulated Swiss 3T3 fibroblasts. This increase is not a result of increased HA synthesis, but is due to a significant increase in the specific activity of the cellular UDP-N-acetyl-glucosamine. When the 3H incorporation is corrected for the change in precursor specific activity, vanadate is shown to inhibit the synthesis of HA, a result that is confirmed by measuring the HA with a competitive binding assay. The inhibition of HA synthesis by vanadate is also shown to be dependent on the media used in the experiment, with a substantial increase in the inhibition seen when the media contains the pH indicator, phenol red. The HA synthetase activity of isolated membranes is not inhibited by vanadate at concentrations up to 500 microM.

Serum stimulation of UDP-glucose dehydrogenase activity in Swiss 3T3 fibroblasts.

A new HPLC assay was developed to measure UDP-glucose dehydrogenase (UDP-GDH) activity in crude homogenates of 3T3 fibroblasts. UDP-GDH activity is directly related to the proliferative activity of the cell culture: enzyme activity is highest in log phase cells and decreases as the culture approaches quiescence. Serum stimulation of quiescent 3T3 fibroblasts results in an increase in UDP-GDH activity that has two components that are differentially affected by inhibitors of protein synthesis. Following serum stimulation, changes in cellular UDP-glucuronic acid concentrations mirror changes in UDP-GDH activity. UDP-xylose is a potent inhibitor of UDP-GDH but inhibitory concentrations of UDP-xylose could not be detected in cell extracts.

Effect of high-protein diet on pyrimidine synthesis and response to PALA in mouse tissues.

BACKGROUND: High-protein diets have been found to protect mice from the lethal effects of cytotoxic pyrimidine analogues and to reduce the toxicity of the antipyrimidine fluorouracil (5-FU), but the biochemical explanation for these effects is not known. PALA potentiates the chemotherapeutic efficacy of 5-FU, and each of the two agents can produce dose-limiting intestinal toxic effects. We have shown that intraperitoneal infusion of ammonium chloride stimulates intestinal de novo pyrimidine synthesis. This stimulation with excess ammonia, which can also result from high-protein intake, is dependent on the presence of carbamoyl phosphate synthetase I, an enzyme in the liver and intestine but not in most tumors. These findings suggest that a high-protein diet can stimulate pyrimidine synthesis in the liver and intestine but leave it unchanged in tumor tissue. PURPOSE: The purpose of this study was to determine whether varying dietary protein causes pharmacologically relevant and preferential changes in de novo pyrimidine synthesis. METHODS: Mice were fed diets containing 18%, 35%, or 50% casein. Dietary effects on de novo pyrimidine synthesis were measured in the intestine, liver, and B16 mouse melanoma in mice treated with PALA and in untreated mice. De novo synthesis was measured by infusion of [15N]alanine into intact animals, determination of 15N incorporation into uracil by use of gas chromatography-mass spectrometry, and calculation of the fraction of the uracil nucleotide pool formed by de novo synthesis. RESULTS: In mice on a 50% casein diet (high protein), de novo pyrimidine synthesis increased substantially in the liver and intestine, compared with synthesis in mice receiving 18% casein. Increase in pyrimidine synthesis in B16 tumor tissue was negligible. The high-protein diet protected the intestine and liver from depletion of uracil nucleotide pools by PALA, and toxicity in tumor-free animals was reduced, as determined by mortality after PALA treatment. Sensitivity of the B16 tumor to the biochemical and cytotoxic effects of PALA was not diminished. CONCLUSIONS: We propose that the basis for these effects of a high-protein diet is the generation of excess carbamoyl phosphate in tissues containing carbamoyl phosphate synthetase I. This carbamoyl phosphate can stimulate de novo pyrimidine synthesis and compete with drugs that interact with enzymes of the de novo pathway, thereby selectively protecting the liver and intestine. IMPLICATIONS: These data provide a biochemical explanation for reported effects of high-protein diet on toxicity of antipyrimidines like 5-FU. Studies are underway to determine if stimulation of pyrimidine synthesis by excess ammonia improves therapy with 5-FU alone or combined with PALA.

Inhibition of fibroblast hyaluronic acid production by suramin.

Hyaluronic acid (HA) is an extracellular matrix glycosaminoglycan localized in the stroma of solid tumors, where it facilitates cell movement and thus tumor invasion and metastasis. This localization of HA is due to its synthesis by stromal fibroblasts in response to paracrine factors produced by the tumor. Such tumor-stromal interactions have been shown to be crucial to the development and progression of prostate cancer. Suramin is an effective antitumor agent in hormone-refractory prostate cancer, but its mechanism(s) of action is not well understood. However, the properties of suramin as an agent which disrupts growth factor action, and the importance of tumor-stroma interactions in prostate tumor development and in HA synthesis led us to study the effect of suramin on HA synthesis. Suramin inhibited HA synthesis by calf serum-stimulated Swiss 3T3 fibroblasts at clinically relevant concentrations (IC50 = 183 micrograms/mL). Increasing the serum concentration from 10 to 20% did not change the IC50 for HA synthesis, but increased the IC50 for [3H]thymidine incorporation from 206 to 342 micrograms/mL, indicating that the antiproliferative effect of suramin can be dissociated from its effect on HA synthesis. Suramin did not alter the cellular concentrations of the two precursors for HA synthesis (UDP-glucuronic acid and UDP-N-acetylglucosamine) at early time points and did not inhibit the HA synthetase activity of isolated membranes at concentrations up to 800 micrograms/mL.(ABSTRACT TRUNCATED AT 250 WORDS)

Contribution of de-novo and salvage synthesis to the uracil nucleotide pool in mouse tissues and tumors in vivo.

The relative contribution of de-novo and salvage synthesis to tissue pyrimidine nucleotide pools is an important parameter in the rational design of anti-pyrimidine therapies, but has not been measured in vivo. We have measured the contribution of de-novo synthesis to the total acid-soluble uracil nucleotide pool in mouse tissues by analysis of the incorporation of label after intra-peritoneal infusion of L-[15N]alanine. The contribution of salvage synthesis was measured by the incorporation of radiolabel after intravenous infusion of [14C]uridine. The results show that de-novo synthesis makes the larger contribution to the intestine uracil nucleotide pool, salvage synthesis makes the larger contribution to the kidney pool, and de-novo and salvage synthesis make roughly equal contributions to the liver pool. In tumors studied (L1210, P388, B16, Nettesheim), the contribution of de-novo synthesis was at least five times the contribution of salvage synthesis. The measurements were repeated 24 hours after a 400-mg/kg dose of N-phosphonacetyl-L-aspartic acid. De-novo synthesis was substantially inhibited in all tissues and tumors after this treatment, although significant residual activity was observed in the intestine and L1210 cells. Nettesheim carcinoma was the only tumor or tissue to show a significant increase in salvage synthesis after N-phosphonacetyl-L-aspartic acid treatment.

De novo synthesis of uracil nucleotides in mouse liver and intestine studied using [15N]alanine.

The amount of newly synthesized uracil nucleotides in mouse liver and intestine was determined by analysis of 15N incorporation into the uracil nucleotide pool of these tissues after intraperitoneal infusion of 15N-labelled amino acids. The appearance of newly synthesized uracil nucleotides was linear with time, and essentially independent of the rate of infusion of L-[15N]alanine. Varying the amino acid used in the infusion could affect the enrichment in the uracil ring nitrogens, but had no significant effect on the calculated amount of de novo synthesis. These results demonstrate the utility of this method in measuring de novo uracil nucleotide synthesis in mouse liver and intestine in vivo. The method should be a valuable tool in the effort to understand the regulation and pharmacological manipulation of de novo uracil nucleotide synthesis.

Rapid increase of cellular UDP-glucuronide after mitogen stimulation of quiescent 3T3 mouse fibroblasts.

Chromatographic analysis of the acid soluble nucleotide pool in 3T3 fibroblasts 30 minutes after serum stimulation revealed one component that was increased compared to quiescent controls. This component was identified as UDP-glucuronide by chromatographic and chemical means. The time course and magnitude of the serum stimulated increase in UDP-glucuronide is similar to the time course and magnitude of the increase in [14C]uridine uptake. Other factors capable of stimulating [14C]uridine uptake, including epidermal growth factor, platelet derived growth factor, interleukin-1, and a phorbol ester also caused an increase in UDP-glucuronide. The results show that one of the earliest changes in pyrimidine nucleotide metabolism after mitogen stimulation is an increase in UDP-glucuronide synthesis, which may be related to increased uridine uptake.

Cellular pharmacology of DUP-785, a new anticancer agent.

DUP-785, a new inhibitor of dihydroorotate dehydrogenase, is currently undergoing clinical evaluation for anticancer activity. We developed a GC/MS method to quantitate dihydroorotate that accumulates in cultures of L1210 cells exposed to growth inhibitory concentrations of DUP-785. This method was used to follow the onset, extent, and duration of inhibition of de novo pyrimidine synthesis in intact L1210 cells and to compare this inhibition with cell proliferation and cellular concentrations of pyrimidine nucleotides. There were direct relations between inhibition of de novo pyrimidine synthesis, changes in pyrimidine nucleotide concentrations, and cell proliferation following short (less than 24 hr) drug exposures; with prolonged exposures (greater than 24 hr), however, there was a departure from these relationships in that restoration of pyrimidine nucleotide pools and de novo pyrimidine pathway activity did not restore cell proliferation. Exposure of L1210 cells to 15 microM DUP-785 produced a maximum cell kill (99.9% as determined by cloning efficiency) at 24 hr, and no increase in cell kill was observed with drug exposure up to 96 hr.

Stimulation of uracil nucleotide synthesis in mouse liver, intestine and kidney by ammonium chloride infusion.

De novo pyrimidine synthesis was studied in mouse liver, intestine, and kidney by intraperitoneal infusion of 15NH4Cl and analysis of 15N incorporation into uracil nucleotide pools. When the dose of a 1-h infusion of 15NH4Cl was increased from 50 mumol to 250 mumol the fraction of the total uracil nucleotide pool formed by de novo synthesis increased 4.0-fold in liver to 8.4% and 2.3-fold in intestine to 13.7%. The increase in intestine was independent of the increase in liver as evidenced by the lack of correlation between the increase observed in the intestine and liver of the same animal and the different distributions of label in the uracil ring nitrogens. A 2.4-fold increase in newly formed uracil nucleotides was observed in kidney when the infusion dose was raised from 150 mumol to 250 mumol. The increase in kidney was correlated with the increase in liver in the same animal and the distribution of label in the uracil ring nitrogens was similar to the distribution in liver. These results suggest that the increase in newly formed uracil nucleotides in intestine is due to increased de novo synthesis of pyrimidines in the intestine, while the increase in the kidney is due to increased salvage synthesis of uracil nucleotides from uridine synthesized in the liver and output to the circulation.

Attempts to increase intratumoral blood flow in the rat solid Walker 256 tumor by the use of the perfluorocarbon emulsion fluosol-DA.

We have examined the effect of the perfluorocarbon emulsion, Fluosol-DA 20% (FDA), on blood flow in rats bearing an advanced solid Walker 256 tumor implanted s.c. Blood-FDA exchange in unanesthetized rats maintained under 100% oxygen was accomplished by simultaneous arterial withdrawal and i.v. infusion until the hematocrit was less than 4%. Control rats were maintained under 100% oxygen but did not undergo any exchange. Regional blood flow studies in tumors of control and FDA-exchanged rats were performed using [14C]iodoantipyrine and quantitative autoradiography. FDA-blood exchange did not increase flow to the whole tumor. Similarly, the pattern of regional flow within the tumor, which was determined in histologically distinct areas--including dense and normocellular, necrotic and peripheral zones invading into muscle and connective tissue--was not substantially altered. Flow to cerebral tissue was increased two-fold, although flow to normal tissues including temporalis muscle, skin and diaphragm was not altered. These results show that FDA-blood exchange does not enhance vascular flow in solid Walker 256 tumor implanted s.c. in the rat.

Increases in brain tumor and cerebral blood flow by blood-perfluorochemical emulsion (Fluosol-DA) exchange.

It has been suggested that oxygen-carrying blood substitutes, perfluorochemical (PFC) emulsions, can increase blood flow and oxygen delivery to poorly perfused tumor regions. Local cerebral blood flow was measured in male Wistar rats bearing intracranial Walker 256 tumor with and without blood-PFC exchange using [14C]iodoantipyrine (IAP) and quantitative autoradiographic techniques. The exchange transfusion was performed in two groups of awake animals breathing 100% oxygen: (a) complete blood-PFC exchange, hematocrit 4%; and (b) partial blood-PFC exchange, hematocrit 20-25%. The tissue/blood partition coefficient for IAP was determined in a separate set of experiments under identical conditions and was used in calculating blood flow. Cerebral blood flow increased approximately 2-fold following complete blood-PFC exchange and 1.5-fold by the partial exchange. A similar 1.5-fold increase in flow was measured in intraparenchymal tumors following partial exchange; however, a flow increase was not identified in the meningeal extension of the tumors. The increase in cerebral blood flow is consistent with an autoregulatory response of the central nervous system vasculature to maintain an adequate supply of oxygen to central nervous system tissue. Presumably, the increase in blood flow to the intracerebral tumor reflects the autoregulatory response of the host tissue. The effect of blood-PFC exchange on blood flow and drug delivery to tumor may depend on the particular tumor and its site of growth (host tissue). The tissue/blood partition coefficient for IAP increased from 0.8 to 1.0 and 1.4 following partial and complete blood-PFC exchange, respectively. This change in the partition coefficient reflects the change in the intravascular fraction of IAP that is bound to plasma proteins. The enhanced therapeutic effect that has been reported in some experimental tumor models may result from a higher tissue/blood equilibrium distribution ratio (due to reduced plasma protein binding) resulting in a higher tissue exposure to certain drugs following PFC administration.

Uracil nucleotide synthesis in a human breast cancer cell line (MCF-7) and in two drug-resistant sublines that contain increased levels of enzymes of the de novo pyrimidine pathway.

Cultured wild-type MCF-7 human breast cancer cells and two MCF-7 sublines that overproduce enzymes of the de novo pyrimidine biosynthetic pathway were compared with regard to: rate of de novo biosynthesis of uracil nucleotides, sensitivity of the de novo and salvage pathways to the concentration of intracellular uracil nucleotides, and potential of exogenous uridine at concentrations equivalent to plasma levels to affect de novo pyrimidine biosynthesis. The PALAR MCF-7 subline, which is resistant to N-(phosphonacetyl)-L-aspartate and has 5.2 times the activity of the first de novo enzyme as the wild-type MCF-7 cells, synthesizes uracil nucleotides via the de novo pathway at a rate that is 5.8 times that of the wild type MCF-7 cells. The PYRR MCF-7 subline, which is resistant to pyrazofurin and has 15.1 times the activity of orotate phosphoribosyltransferase as the wild-type MCF-7 cells, synthesizes uracil nucleotides via the de novo pathway at a rate that is 1.4 times that of wild-type MCF-7 cells. These results are consistent with carbamyl phosphate synthetase being the rate-controlling step of de novo pyrimidine biosynthesis. In the presence of exogenous uridine at concentrations equivalent to that found in plasma (4.4-8.6 microM), the uracil nucleotide pool of wild-type MCF-7 cells was expanded by 20% and de novo synthesis was inhibited by 55%. Incubation of PALAR MCF-7 cells with uridine at concentrations between 7.3 and 16.8 microM caused a 40% increase in the uracil nucleotide pool and a 30% inhibition of de novo synthesis. De novo synthesis of uracil nucleotides in PYRR MCF-7 cells was not affected by a greater than 10-fold increase in the uracil nucleotide pool. Salvage of [14C] uridine was inhibited by an expanded uracil nucleotide pool in the wild-type and PYRR MCF-7 cells but was not inhibited in the PALAR MCF-7 cell line. These results demonstrate that, although the overproduced enzymes exhibit substrate affinities and specificities in cell-free preparations similar to those of the wild-type enzymes, in intact cells the resistant cell lines exhibit marked differences in the control of de novo and salvage pyrimidine biosynthetic pathways by intracellular uracil nucleotides.

Adenosine export from the liver: oxygen dependency.

The liver is believed to be an important regulator of purine concentrations in the circulation. It has been an accepted notion that the liver exports the riboside adenosine to the circulation for use by other tissues. This phenomenon was reexamined using an artificial oxygen carrier to perfuse the isolated rat liver and high-performance liquid chromatography (HPLC) to analyze the purine content. It was found that perfusion with Krebs-Ringer-bicarbonate at a physiological flow rate as had been used by previous investigators results in a mildly hypoxic preparation, and it is this hypoxia that results in the export of adenosine. In a liver perfused with Fluosol-43, little (less than 0.05 microM) or no adenosine was released. However, if the oxygen content of Fluosol-43 was decreased, the liver could be induced to release adenosine at 1.0 microM levels. In addition, the liver was found to release adenine under control conditions; thus it is this purine base that is likely to be the important purine released by the liver for extrahepatic salvage, not adenosine as previously thought.

The effect of 3-deazauridine and dipyridamole on uridine utilization by mice.

The inhibition of uridine utilization by 3-deazauridine, an inhibitor of uridine kinase, and by dipyridamole, an inhibitor of the facilitated transport of nucleosides was examined. 3-Deazauridine (500 mg/kg) markedly inhibited (greater than 70%) the formation of uracil nucleotides from uridine in liver, kidney, and L1210 tumor cells. The degree of inhibition is greatly reduced by 6 hr after administration of the drug. Dipyridamole (100 mg/kg) did not significantly reduce salvage of uridine by liver or kidney and produced only small, transient reductions in salvage by L1210 tumors. Dipyridamole pretreatment did not alter the rate of clearance of uridine from the plasma.