A critical role for uridine nucleotides in the regulation of deoxycytidine kinase and the concentration dependence of 1-beta-D-arabinofuranosylcytosine phosphorylation in human leukemia cells

The intracellular concentration of 1-beta-D-arabinofuranosylcytosine (ara-C) for half-maximal phosphorylation by leukemic blasts obtained directly from patients was 2.1 +/- 2.5 microM (median, 1.3 microM, N = 25), and the rate of ara-C accumulation actually declined at concentrations above 20 microM in 35% of these cell populations. These apparent Km values for cellular phosphorylation were an order of magnitude lower than the Km of deoxycytidine (dCyd) kinase for ara-C with ATP as phosphate donor. dCyd kinase was purified from human leukemia cells and assayed for [3H]ara-C kinase activity with a mixture of 7 nucleotides at their approximate cellular concentrations or with a single nucleotide deleted. At low or high ara-C concentrations, ATP, GTP, CTP, or dTTP could be eliminated without significantly altering the rate. The only potential phosphate donor that was clearly important was UTP, since its deletion reduced the rate to only 25% of that with the complete mix. As anticipated, eliminating dCTP, the end product of this salvage pathway, moderately increased the rate by 50% at 0.4 microM ara-C or by 26% at 40 microM ara-C. At 40 microM ara-C, deleting UDP from the mix increased the rate more than deleting dCTP. dCTP was less inhibitory against 1 mM UTP (50% inhibitory concentration, 26 microM) than against 4 mM ATP (50% inhibitory concentration, 2.2 microM). In kinetic assays with 4 mM ATP and variable ara-C, UDP was a potent uncompetitive inhibitor with a Ki of 4 microM; the Ki for ADP was 1000-fold higher. Direct fit of kinetic data to the Michaelis equation yielded a Km for ara-C of 49 microM with 4 mM ATP as the phosphate donor; however, there was evidence of negative cooperativity with a Hill coefficient of 0.7. High ara-C Km values were also obtained with GTP and CTP, but with no evidence of cooperativity. With 1 mM UTP, the Km was 1.5 microM with moderate substrate inhibition; thus the kinetic data with UTP were similar to those for ara-C phosphorylation by intact cells. UDP was less potent versus UTP than versus ATP. It lowered the Vmax and enhanced the ara-C substrate inhibition without altering the Km. When 1 mM UTP and 4 mM ATP were mixed, the kinetic pattern was similar to that for UTP alone. The Km for UTP with [3H]dCyd as the phosphate acceptor of 0.8 microM was 25-fold lower than the Km for ATP of 20 microM.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sensitization of ara-C-resistant lymphoma cells by a pronucleotide analogue

Adequate intracellular concentrations of ara-CMP, the monophosphorylated derivative of ara-C, are essential for its cytotoxicity. The critical step for ara-CMP formation is intracellular phosphorylation of ara-C by deoxycytidine kinase (dCK). A common nucleoside resistance mechanism is mutation affecting the expression or the specificity of dCK. We describe the ability of a tert-butyl S-acyl-thioethyl (SATE) derivative of ara-CMP (UA911) to circumvent ara-C resistance in a dCK-deficient human follicular lymphoma cell line (RL-G). The RL-G cell line was produced by continuous exposure to gemcitabine and displayed low dCK mRNA and protein expression that conferred resistance both to ara-C (2,250-fold) and to gemcitabine (2,092-fold). RL-G cells were able to take up the UA911 pronucleotide by diffusion and metabolize it to the corresponding ara-CMP and ara-CTP nucleotides, exhibiting a 199-fold reduction in resistance ratios, and a similar cell cycle arrest to the parental RL-7 cells. Exposures to 10, 50 or 100 microM concentrations of UA911 produced 160 +/- 7, 269 +/- 8 and 318 +/- 62 pmol ara-CTP/mg protein in RL-7 cells, and 100 +/- 12, 168 +/- 10 and 217 +/- 39 pmol ara-CTP/mg protein in RL-G cells, respectively. Exposure of RL-G cells to underivatized, radiolabeled ara-C produced no detectable amounts of the active triphosphate metabolites. We conclude that the UA911 pronucleotide is capable of overcoming dCK-mediated resistance. This result can be attributed to the unique cellular metabolism of the SATE pronucleotides giving rise to the intracellular delivery of ara-CMP to dCK-deficient cells.     

The effect of edelfosine on CTP: Cholinephosphate cytidylyltransferase activity in leukemic cell lines

Analogs of ether phospholipids have been shown to have selective anti-neoplastic activity. The compounds are known to inhibit phospholipid biosynthesis. This paper examines the effect of the alkyl-lysophospholipid, edelfosine, on the rate-limiting enzyme, CTP:choline-phosphate cytidylyltransferase, in de novo phosphatidylcholine synthesis in sensitive and resistant leukemic cell lines. Enzyme activity was measured by the incorporation of ¹⁴C-phosphocholine into CDP-choline by lysates of HL60 and K562; cells demonstrated inhibition of incorporation of ¹⁴C-phosphocholine in HL60 cell lysates but no inhibition in K562 lysates.Partial purification of cytidylyltransferase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting demonstrated similarity between the enzyme isolated from each cell line. Cloning and sequencing of cytidylyltransferase cDNA of HL60 cells was accomplished using a probe encoding the entire protein sequence of the K562 cytidylyltransferase gene. A substitution at nucleotide 751 from A in the HL60 cell cDNA clone to G in the K562 cDNA clone resulted in a change in amino acid number 251 from lysine (positively charged) in the HL60 enzyme to glutamic acid (negatively charged) in the K562 enzyme. This negative charge in the lipid-binding domain of the K562 enzyme may result in a weaker binding of edelfosine and the observed decrease in activity, as evidenced by resistance to edelfosine by K562 cells.     

Biotransformation of aflatoxin B1 in rabbit lung and liver microsomes

Aflatoxin B1 (AFB1) is a potent hepatotoxic and hepatocarcinogenic mycotoxin that requires bioactivation to AFB1-2,3-oxide for activity. In addition to epoxidation, microsomal monooxygenases biotransform AFB1 to the less toxic metabolites, aflatoxin M1 (AFM1) and aflatoxin Q1 (AFQ1). The lung is at risk from AFB1 both via inhalation and via the circulation. In the present study, we have characterized rabbit lung and liver microsomal AFB1-DNA binding (an index of AFB1-2,3-oxide formation), AFM1 formation and AFQ1 formation. Vmax values for AFB1-DNA binding were not different between lung and liver when expressed per mg microsomal protein (1.06 +/- 0.13 and 2.12 +/- 1.30 nmol/mg/h for lung and liver respectively), but lung values were greater than liver when expressed per nmol cytochrome P450 (3.64 +/- 0.31 and 1.29 +/- 0.70 nmol/nmol P450/h for lung and liver respectively). Km values for this reaction were not different between lung and liver. Vmax values for AFM1 formation in liver microsomes were greater than in lung when expressed per mg protein, but not when expressed per nmol P450. No differences were detected for the Km for AFM1 formation between lung and liver microsomes. For AFQ1 formation, no differences were detected between Vmax values of lung and liver, regardless of whether results were expressed per mg protein or per nmol P450, while the Km for AFQ1 formation was lower in liver. SKF-525A inhibited these reactions by 63-74% in lung microsomes and 90-96% in liver microsomes. These results indicate that the lung is capable of activating AFB1, and that rabbit lung microsomes contain high activity for this reaction. Furthermore, little AFM1 and AFQ1 are formed in lung microsomes, leading to minimal shunting of AFB1 from the activation pathway.     

Antitumor activity of 1-beta-D-arabinofuranosylcytosine conjugated with polyglutamic acid and its derivative

Four types of 1-beta-D-arabinofuranosylcytosine (ara-C) conjugates with poly-L-glutamic acid (PLGA) or poly-N5-(2-hydroxyethyl)-L-glutamine (PHEG) were prepared in an attempt to enhance the efficacy of the drug in simple dosage schedules. The conjugates were made by linking ara-C to the carboxyl groups of PLGA directly at N-4 of ara-C (ara-C:PLGA) or indirectly through the 2-aminoethylphosphoryl or 6-aminohexylphosphoryl side chain which had been introduced to C-5' of ara-C, 1-[5'-(2-aminoethylphosphoryl)-beta-D-arabinofuranosyl]cytosine: PLGA [araCMP(C2):PLGA and 1-[5'-(6-aminohexylphosphoryl)-beta-D-arabinofuranosyl]cytosine:++ +PLGA, respectively, or made by converting the remaining carboxyl groups in the PLGA conjugates to the 2-hydroxyethylamide groups [ara-C:PHEG, ara-CMP(C2):PHEG, 1-[5'-(6-aminohexylphosphoryl)-beta-D-arabinofuranosyl]cytosine:++ +PHEG]. Studies in vitro showed that the conjugates had decreased cytotoxicity against L1210 cells when compared with that of ara-C. Studies in vivo showed that all of the conjugates, except ara-CMP(C2):PLGA, had a greater antitumor activity than did ara-C in L1210 tumor-bearing BALB/c X DBA/2 F, (hereafter called CD2F1) mice (inoculum, 1 X 10(5) cells i.p. on Day 0) which were treated by a single i.p. injection of either the conjugates or the control ara-C on Day 1. The largest antitumor activity [increased life span (ILS) 170%] was observed with a dosage of 50 mg (equivalent ara-C per kg) of ara-C:PHEG. When CD2F1 mice which had been inoculated i.p. with 1 X 10(5) L1210 cells were treated with an i.p. injection of 12.5 or 25 mg (equivalent ara-C per kg) of ara-C:PHEG daily for 5 days starting from Day 1, 2 of 5 mice survived more than 42 days, and the ILS of the remaining mice was 153 and 184%. The injections of 3.2 mg (equivalent ara-C per kg) of ara-C:PHEG showed a moderate antitumor activity with an ILS of 113% which was similar to the ILS (119%) found when unconjugated ara-C (400 mg/kg) was used to treat tumor-bearing mice. In in vitro release experiments, ara-C was released slowly from ara-C:PLGA at pH 7.4, and ara-CMP(C2):PLGA was chemically stable but cleaved by phosphodiesterase, acid phosphatase, and alkaline phosphatase to give mainly 1-beta-D-arabinofuranosylcytosine 5'-monophosphate.     

Molecular mechanisms involved in farnesol-induced apoptosis

The isoprenoid alcohol farnesol is an effective inducer of cell cycle arrest and apoptosis in a variety of carcinoma cell types. In addition, farnesol has been reported to inhibit tumorigenesis in several animal models suggesting that it functions as a chemopreventative and anti-tumor agent in vivo. A number of different biochemical and cellular processes have been implicated in the growth-inhibitory and apoptosis-inducing effects of farnesol. These include regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and CTP:phosphocholine cytidylyltransferase α (CCTα), rate-limiting enzymes in the mevalonate pathway and phosphatidylcholine biosynthesis, respectively, and the generation of reactive oxygen species. In some cell types the action of farnesol is mediated through nuclear receptors, including activation of farnesoid X receptor (FXR) and peroxisome proliferator-activated receptors (PPARs). Recent studies have revealed that induction of endoplasmic reticulum (ER) stress and the subsequent activation of the unfolded protein response (UPR) play a critical role in the induction of apoptosis by farnesol in lung carcinoma cells. This induction was found to be dependent on the activation of the MEK1/2-ERK1/2 pathway. In addition, farnesol induces activation of the NF-κB signaling pathway and a number of NF-κB target genes. Optimal activation of NF-κB was reported to depend on the phosphorylation of p65/RelA by the MEK1/2-MSK1 signaling pathway. In a number of cells farnesol-induced apoptosis was found to be linked to activation of the apoptosome. This review provides an overview of the biochemical and cellular processes regulated by farnesol in relationship to its growth-inhibitory, apoptosis-promoting, and anti-tumor effects.     

A novel in vitro model system for studying the action of ara-C

 The antimetabolite 1-β-D-arabinofuranosylcytosine (ara-C) has proven to be one of the most effective agents available for the treatment of acute leukemia. While ara-C has been implicated as a potent inhibitor of mammalian cell DNA replication, the specific mechanism by which ara-C kills cells is not known. In this report we describe the development of an in vitro model system to study the molecular mechanism of ara-CMP incorporation into DNA. This model system makes use of a recently described human cell multiprotein DNA replication complex (MRC) that is competent to replicate DNA in vitro. The MRC can successfully incorporate ara-CMP into replicating DNA at internucleotide positions. These results are similar to those described for studies using intact cells. This MRC-driven in vitro replication system may therefore serve as a powerful model for the study of anticancer agents that directly affect human cell DNA synthesis. Received: 31 March 1995/Accepted: 9 October 1995     

Deuterium isotope effect on denitrosation and demethylation of N-nitrosodimethylamine by rat liver microsomes

In an attempt to elucidate the molecular basis for the decrease in rat liver carcinogenicity and DNA-alkylating ability that accompanies deuteration of N-nitrosodimethylamine (NDMA), NDMA and its fully deuterated analogue ([2H6]NDMA) were incubated with acetone-induced rat liver microsomes. Rates for the competing metabolic routes, denitrosation and demethylation, were determined from colorimetric data on nitrite and formaldehyde generation, respectively. The Vmax calculated for demethylation of NDMA was 7.9 nmol/min/mg, while that for denitrosation was 0.83 nmol/min/mg. Deuteration of NDMA did not significantly change the Vmax for either pathway, but it did increase the Km for demethylation from 0.06 to 0.3 mM. The Km for denitrosation was also increased from 0.06 to 0.3 mM on deuteration, as determined by incubating an equimolar mixture of amino-15N-labeled NDMA with [2H6]NDMA and measuring the methyl[15N]amine:[2H3]methylamine ratio by derivatization-gas chromatography-mass spectrometry. The fact that the Km values for denitrosation were so similar to those for demethylation suggested that the two pathways were catalyzed by the same enzyme. The isotope effects calculated from these data [VmaxH/VmaxD approximately 1 and (Vmax/Km)H/(Vmax/Km)D approximately 5] show that microsomal metabolism of NDMA is not significantly shifted from demethylation to denitrosation on deuteration of substrate and may indicate a low commitment to catalysis for the enzyme. The results are consistent with the view that the metabolism of NDMA is initiated by formation of an alpha-nitrosamino radical which either combines with a hydroxyl radical to form the alpha-hydroxynitrosamine as the initial product of the demethylation pathway or fragments to nitric oxide and N-methylformaldimine as the first products of denitrosation.     

Alteration of the pharmacokinetics of high-dose ara-C by its metabolite, high ara-U in patients with acute leukemia

The pharmacokinetics of high-dose cytosine arabinoside (HiDAC) given as a three-hour intravenous infusion at 3 g/m2 were studied in five patients with acute leukemia during relapse and/or remission of their disease. Apparent steady state plasma levels of ara-C during 13 infusions averaged 115 +/- 32 microM. Upon cessation of the infusion, cytosine arabinoside (ara-C) was rapidly cleared from the plasma. The apparent postinfusion kinetics of ara-C were triexponential with a distribution half-life of 16 minutes and elimination half-lives of 1.8 hours and six hours. Total clearance averaged 86 L per hour and mean residence time averaged 0.47 hours. Disease status (relapse or remission) had no apparent effect on the pharmacokinetic characteristics of ara-C. Peak levels of ara-U averaged 310 microM and the metabolite had an average apparent elimination half-life of 3.75 hours. Despite the persistence of ara-U at about 100 microM at the time of administration of subsequent infusions of ara-C, there was no further accumulation of ara-U in the plasma with repetitive infusions of HiDAC. In vitro studies indicate that ara-U can exert an inhibitory effect on deoxycytidine (dCyd) deaminase activity. The ratio of the Ki of ara-U to the Km of ara-C for cytidine (Cyd)-dCyd deaminase is 40:1; however, during the gamma phase of ara-C elimination, the ratio of ara-U:ara-C in plasma is at least 100:1. Thus, a retardation of systemic catabolism of ara-C by ara-U is possible. Two to three hours after the termination of the HiDAC infusion, the ara-C cerebrospinal fluid: plasma ratio is 1-3:1, a feature of potential therapeutic significance. The slower elimination of ara-C from the CSF may also contribute to the plasma gamma half-life.     

Regulation of the cytidine phospholipid pathways in human cancer cells and effects of 1-beta-D-arabinofuranosylcytosine: a noninvasive 31P nuclear magnetic resonance study

Using 31P nuclear magnetic resonance spectroscopy we have noninvasively observed metabolic control through the cytidine pathways of phosphatidylcholine and phosphatidylethanolamine synthesis in intact actively metabolizing MDA-MB-231 human breast cancer cells. Perfusion with the phospholipid precursors ethanolamine or choline (2 mM) indicates that the cytidylyltransferase enzymes are rate limiting for both pathways. Complete inhibition of choline kinase with ethanolamine allowed the observation of the utilization of phosphocholine by the rate-limiting enzyme choline-phosphate cytidylyltransferase. The rate was dependent on the phosphocholine concentration. Inhibition of glycerophosphorylcholine phosphodiesterase with accumulation of substrate was also observed and allows an estimate of the flux through the degradative pathways. The human lymphoma cell line MOLT-4 was also found to contain high levels of phosphocholine and phosphoethanolamine. The levels of these precursors in the MOLT-4 line are lowered by 40% after 6 h when perfused with high dose 1-beta-D-arabinofuranosylcytosine (Ara-C) (400 microns) but are unaffected by 2 microns Ara-C or dideoxycytidine. High dose Ara-C also resulted in lysis in 8-10 h. However, the MDA-MB-231 cell line which is not sensitive to Ara-C showed no change in its spectrum when perfused with Ara-C. A potential mechanism based on classic phospholipid metabolism for the lytic effect of high dose Ara-C is discussed.     

Gemcitabine diphosphate choline is a major metabolite linked to the Kennedy pathway in pancreatic cancer models in vivo

Background:

The modest benefits of gemcitabine (dFdC) therapy in patients with pancreatic ductal adenocarcinoma (PDAC) are well documented, with drug delivery and metabolic lability cited as important contributing factors. We have used a mouse model of PDAC: KRAS^G12D; p53^R172H; pdx-Cre (KPC) that recapitulates the human disease to study dFdC intra-tumoural metabolism.

Methods:

LC-MS/MS and NMR were used to measure drug and physiological analytes. Cytotoxicity was assessed by the Sulphorhodamine B assay.

Results:

In KPC tumour tissue, we identified a new, Kennedy pathway-linked dFdC metabolite (gemcitabine diphosphate choline (GdPC)) present at equimolar amounts to its precursor, the accepted active metabolite gemcitabine triphosphate (dFdCTP). Utilising additional subcutaneous PDAC tumour models, we demonstrated an inverse correlation between GdPC/dFdCTP ratios and cytidine triphosphate (CTP). In tumour homogenates in vitro, CTP inhibited GdPC formation from dFdCTP, indicating competition between CTP and dFdCTP for CTP:phosphocholine cytidylyltransferase (CCT). As the structure of GdPC precludes entry into cells, potential cytotoxicity was assessed by stimulating CCT activity using linoleate in KPC cells in vitro, leading to increased GdPC concentration and synergistic growth inhibition after dFdC addition.

Conclusions:

GdPC is an important element of the intra-tumoural dFdC metabolic pathway in vivo.     

Alterations of choline phospholipid metabolism in ovarian tumor progression

Recent characterization of abnormal phosphatidylcholine metabolism in tumor cells by nuclear magnetic resonance (NMR) has identified novel fingerprints of tumor progression that are potentially useful as clinical diagnostic indicators. In the present study, we analyzed the concentrations of phosphatidylcholine metabolites, activities of phosphocholine-producing enzymes, and uptake of [methyl-14C]choline in human epithelial ovarian carcinoma cell lines (EOC) compared with normal or immortalized ovary epithelial cells (EONT). Quantification of phosphatidylcholine metabolites contributing to the 1H NMR total choline resonance (3.20-3.24 ppm) revealed intracellular [phosphocholine] and [total choline] of 2.3 +/- 0.9 and 5.2 +/- 2.4 nmol/10(6) cells, respectively, with a glycerophosphocholine/phosphocholine ratio of 0.95 +/- 0.93 in EONT cells; average [phosphocholine] was 3- to 8-fold higher in EOC cells (P < 0.0001), becoming the predominant phosphatidylcholine metabolite, whereas average glycerophosphocholine/phosphocholine values decreased significantly to < or =0.2. Two-dimensional (phosphocholine/total choline, [total choline]) and (glycerophosphocholine/total choline, [total choline]) maps allowed separate clustering of EOC from EONT cells (P < 0.0001, 95% confidence limits). Rates of choline kinase activity in EOC cells were 12- to 24-fold higher (P < 0.03) than those in EONT cells (basal rate, 0.5 +/- 0.1 nmol/10(6) cells/h), accounting for a consistently elevated (5- to 15-fold) [methyl-14C]choline uptake after 1-hour incubation (P < 0.0001). The overall activity of phosphatidylcholine-specific phospholipase C and phospholipase D was also higher ( approximately 5-fold) in EOC cells, suggesting that both biosynthetic and catabolic pathways of the phosphatidylcholine cycle likely contribute to phosphocholine accumulation. Evidence of abnormal phosphatidylcholine metabolism might have implications in EOC biology and might provide an avenue to the development of noninvasive clinical tools for EOC diagnosis and treatment follow-up.     

Modulation of the cellular pharmacokinetics of ara-CTP in human leukemic blasts by dipyridamole

Summary The effect of dipyridamole (DP) on the cellular retention of 1--d-arabinofuranosylcytosine (ara-C) and its metabolites was examined in leukemic blasts that had been isolated directly from bone marrow aspirates from patients afflicted with acute myeloid leukemia (AML). When AML cells were loaded for 2 h with 1 m [³H]-ara-C and then transferred to ara-C-free medium, total intracellular concentrations of radiolabel and [³H]-ara-C 5-triphosphate [³H]-ara-C-CTP rapidly declined. After 8 h, total intracellular levels of tritium were 4.4 times higher if 10 m was included in the washout medium; however, the majority of this intracellular radiolabel corresponded to [³H]-uridine arabinoside ([³H]-ara-U) and [³H]-ara-C. DP significantly increased the meant _1/2 for [³H]-ara-CTP from 102 to 136 min (P<0.01), but this effect was much less pronounced than that obtained for total tritium and the increase was quite variable (0–70%; median, 19%). The presence of DP in the washout medium also increased the incorporation of ara-C into DNA and the formation of ara-CDP-choline. The level of ara-CDP-choline continued to increase in both DP-containing and DP-free media for the first 4 h following drug removal and the formation of ara-CDP-choline continued during the first few hours in ara-C-free medium. At the end of the 8-h wash in DP-containing medium, the cellular concentration of ara-CDP-choline was equivalent to that found at the beginning of the washout period. Although statistically significant, the effect of DP on ara-CTP retention in AML blasts was much less pronounced than that previously observed in L5178Y leukemia. The former cells exhibited only 10% as many nucleoside transport carriers as did the L5178Y cells as measured by their capacity to bind [³H]-nitrobenzylmer-captopurine riboside (NBMPR). The effect of DP in prolonging ara-CTP retention was proportional to the number of [³H]-NBMPR binding sites. This suggests that in patients cells that exhibit extremely low transport capacity, most of the net catabolism occurs via deamination, and further inhibition of transport by DP in an effort to improve cellular retention of ara-C has little effect on ara-CTP catabolism.Abbreviations ara-C 1--d-Arabinofuranosylcytosine, cytosine arabinoside - ara-CMP ara-C 5-monophosphate - ara-CTP ara-C 5-triphosphate - ara-C-DNA ara-C incorporated into DNA - DP dipyridamole - Persantine 2,2,2,2-[4,8-dipiperidinopyrimido(5,4-d)-pyrimidine-2,6-dinitrilo] tetraethanol - NBMPR nitrobenzylmercaptopurine riboside Present address:US Bioscience Inc., One Tower Bridge, 100 Front Streen, West Conshohocken, PA 19428, USASupported by grant CH-471L from the American Cancer Society. This work was also supported in part by the Leukemia Cell Distribution Laboratory of the Comprehensive Cancer Center of Wake Forest University (grant CA 12 197). One of the authors (J. C. W.) is a Leukemia Society of America Scholar     

The transport of pteridines in CCRF-CEM human lymphoblastic cells

The transport routes used by CCRF-CEM human lymphoblastoid cells for the influx and efflux of unconjugated pteridines were analyzed using [3H]6-hydroxymethylpterin as a model compound. Influx proceeds by a mechanism that exhibits a Km of 66.7 microM and a Vmax of 0.077 nmol/min per mg cellular protein. The process is somewhat sensitive to metabolic inhibitors, particularly uncouplers of oxidative phosphorylation, and is significantly affected by the presence of other pteridines in the extracellular medium. The results suggest that pterins with either no 6-substituent (pterin) or those with methyl, hydroxyl, or formyl groups in this position, which exhibit Ki values between 25 and 77 microM, may share the same pathway for uptake. 6-Carboxypterin exhibits low affinity for the system (Ki greater than 500 microM), as do 7-substituted and 6,7-di-substituted derivatives and compounds with larger groups at the 6-position, such as neopterin and biopterin (Ki = 250-300 microM). Efflux of [3H]6-hydroxymethylpterin occurs rapidly and can proceed by at least two routes. The first, comprising approximately 50% of total efflux, is inhibited by extracellular pterins and exhibits similar properties to the uptake system in both its pattern of sensitivity to metabolic inhibitors and its specificity for pteridine structure. The route by which the remaining efflux occurs is relatively insensitive to metabolic inhibition. Adenine significantly inhibits 6-hydroxymethylpterin influx and efflux (Ki = 10.6 microM for uptake) but does not appear to share the same transport system. Similarly, methotrexate and folic acid exhibit little affinity for the unconjugated pteridine transport routes.     

All-trans-retinoic acid increases cytosine arabinoside cytotoxicity in HL-60 human leukemia cells in spite of decreased cellular ara-CTP accumulation

Background: Accumulation of the cytosine arabinoside (ara-C) metabolite ara-C-triphosphate (ara-CTP) in leukemic blast cells is considered to be the main determinant of ara-C cytotoxicity in vitro and in vivo. Retinoids such as all-trans-retinoic acid (ATRA) have been shown to increase the sensitivity of acute myelogenous leukemic (AML) blast cells to ara-C. To investigate the mechanism of this sensitisation, the hypothesis was tested that ATRA augments cellular ara-CTP levels in human-derived myelogenous leukemia HL-60 cells.Materials and methods: The effect of ATRA and 13-cis-retinoic acid on ara-CTP accumulation and ara-C-induced apoptosis was studied. Ara-CTP levels were measured by high-performance liquid chromatography (HPLC), cytotoxicity by the tetrazolium (MTT) assay, and apoptosis by occurrence of DNA fragmentation (gel electrophoresis), cell shrinkage and DNA loss (flow cytometry).Results: Pretreatment of HL-60 cells with ATRA (0.01–1 µM) caused a significant decrease in intracellular ara-CTP levels; e.g., incubation for 72 hours with ATRA 1 µM prior to one hour ara-C 10 µM reduced ara-CTP levels to 41% ± 4% of control. Similar results were obtained after preincubation with 13-cis-retinoic acid. In spite of decreased ara-CTP levels, the cytotoxicity of the combination was supraadditive and ATRA augmented ara-C-induced apoptosis.Conclusions: At therapeutically relevant concentrations ATRA increased ara-C cytotoxicity and ara-C induced apoptosis but this augmentation is not the corollary of elevated ara-CTP levels. The feasibility of ara-C treatment optimisation via strategies other than those involving elevation of ara-CTP levels should be investigated further.     

Transport of amino acid amide sarcosinamide and sarcosinamide chloroethylnitrosourea in human glioma SK-MG-1 cells.

The transport of the amino acid amide N-[3H]sarcosinamide (methyl glycinamide) was investigated in human glioma SK-MG-1 cells. Sarcosinamide uptake was found to be temperature dependent, sodium independent, and linear up to 1 min at 22 degrees C. Equilibrium was reached after 10 min at 22 degrees C with accumulation slightly above unity. Sarcosinamide was not metabolized in the cells as shown by thin layer chromatography. The uptake of sarcosinamide was significantly decreased when the extracellular pH was lowered from 7.5 to 6.0 and significantly enhanced at pH values above 7.5. The latter effect may be due mainly to increased cell permeability at high pH. The uptake of the labeled sarcosinamide was trans-stimulated by excess cold sarcosinamide. Sarcosinamide uptake over a 200-fold range of concentrations followed Michaelis-Menten kinetics with a Km of 0.284 +/- 0.041 mM and a Vmax of 0.154 +/- 0.024 nmol/10(6) cells/min. The uptake of sarcosinamide was significantly reduced by iodoacetate but not by the metabolic poisons NaF, ouabain, or dinitrophenyl, suggesting that the uptake is not dependent on energy, rather it proceeds by facilitated diffusion. Several naturally occurring substrates were unable to inhibit the uptake of sarcosinamide. Leucine significantly reduced the uptake of sarcosinamide, while sarcosinamide was a weak inhibitor of leucine transport. 2-Aminobicyclo[2,2,1]heptane-2-carboxylic acid a specific substrate for the sodium-independent, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid-sensitive amino acid system L failed to inhibit the uptake of sarcosinamide. Epinephrine reduced the uptake of sarcosinamide and sarcosinamide was equally potent as an inhibitor of epinephrine transport. Dixon plot analysis demonstrated that epinephrine (Km = 0.270 mM) inhibits the uptake of sarcosinamide competitively (Ki = 0.260 mM). These results indicate that sarcosinamide is a substrate for the catecholamine transporter. The alkylating agent, sarcosinamide chloroethylnitrosourea, was tested for its ability to inhibit the uptake of sarcosinamide. The results of Dixon plot analysis were consistent with competitive inhibition of sarcosinamide uptake and the inhibition constant Ki for SarCNU was found to be 3.26 +/- 0.57 mM. The steady-state intracellular concentration of SarCNU was found to be significantly higher (cell:medium ratio of 1.03 +/- 0.01) than that of BCNU cell:medium ratio of 0.52 +/- 0.12). These findings indicate that SarCNU and sarcosinamide share the same carrier for uptake in SK-MG-1 cells. This transport mechanism may be responsible for the increased accumulation of SarCNU as compared to BCNU, a nitrosourea which enters cells by passive diffusion.     

IMP dehydrogenase: inhibition by the anti-leukemic drug, tiazofurin

Tiazofurin through its active metabolite thiazole-4-carboxamide adenine dinucleotide (TAD) inhibits IMP dehydrogenase, the rate-limiting enzyme of GTP biosynthesis. IMP dehydrogenase activity in human leukemic cell extracts (33.4 +/- 0.1 nmol/h/mg protein) was increased 11-fold compared to normal leukocytes (3.1 +/- 0.5). Km values for IMP and NAD+ of leukemic IMP dehydrogenase were 22.7 and 44.0 microM, respectively. XMP inhibited competitively with IMP and noncompetitively with NAD+. NADH exerted mixed type inhibition with respect to both IMP and NAD+. The inhibitory pattern of TAD was quite similar to that of NADH; however, the affinity of TAD to leukemic IMP dehydrogenase (Ki = 0.1 microM) was three orders of magnitude higher than the natural product NADH (Ki = 150 microM). These results contribute to an understanding of the mechanism of action of tiazofurin in the treatment of leukemia.     

Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by human cytochrome P4501A1, P4501A2 and P4501B1

While the metabolic activation of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) by N-hydroxylation has been well documented, the relative roles of the human cytochrome P450 (CYP) enzymes that catalyze this reaction have not been established. Previous studies indicated that the mutagenic activation product, 2-hydroxyamino-PhIP (N2-OH- PhIP), is produced primarily by CYP1A2, and to a lesser extent by CYP1A1. We recently reported that human CYP1B1 also produces N2-OH-PhIP (Carcinogenesis, 18, 1793-1798, 1997). In the present study, we examined PhIP metabolism by microsomes containing recombinant human CYP1A1, 1A2 or 1B1 expressed in Sf9 insect cells and compared the kinetic values for PhIP metabolite formation. PhIP metabolites were analyzed by high pressure liquid chromatography with fluorescence and absorbance detection. Vmax values for N2-OH-PhIP formation were 90, 16 and 0.2 nmol/min/nmol P450, and the apparent Km values were 79, 5.1 and 4.5 microM for human CYP1A2, 1A1 and 1B1, respectively. The non- mutagenic metabolite, 4'-hydroxy-PhIP, was also formed by all three CYP enzymes with Vmax values of 1.5, 7.8 and 0.3 nmol/ min/nmol P450 and apparent Km values of 43, 8.2 and 2.2 microM for human CYP1A2, 1A1 and 1B1, respectively. Although the Vmax for N2-OH-PhIP production was highest for CYP1A2, the catalytic efficiency (Vmax/Km) of CYP1A1 was greater than that of CYP1A2. These results suggest that, for humans, extrahepatic CYP1A1 may be more important than previously thought for the metabolic activation of the dietary carcinogen PhIP.      

Inhibition of nucleoside transport in murine lymphoma L5178Y cells and human erythrocytes by the uridine phosphorylase inhibitors 5-benzylacyclouridine and 5-benzyloxybenzylacyclouridine

The uridine phosphorylase inhibitors, 5-benzylacyclouridine (BAU) and 5-benzyloxybenzylacyclouridine (BBAU) (Biochem. Pharmacol., 31: 1857, 1982), inhibited uptake of uridine in L5178Y cells. By a rapid sampling technique, BAU and BBAU were shown to inhibit the transport (zero-trans influx) of uridine, thymidine, and adenosine in human erythrocytes as well as in murine L5178Y cells. In all cases, competitive inhibitions were observed. Km values for the transport of adenosine, uridine, and thymidine in erythrocytes were 2.2, 195, and 199 microM, while Vmax were 2.9, 118, and 96.5 pmol/min/10(6) cells, respectively. In L5178Y cells, Km values of 14.8 and 23.1 microM and Vmax of 389 and 176 pmol/min/10(6) cells were obtained for adenosine and uridine, respectively. For erythrocytes, the Ki values of BAU were 127, 124, and 198 microM using adenosine, uridine, and thymidine as the substrate; and those of BBAU, 14.1 and 19.2 microM for adenosine and uridine, respectively. In L5178Y, the Ki values of BAU were 202 and 234 microM, and those of BBAU, 39.8 and 27.9 microM for adenosine and uridine, respectively. These data indicate that, in two cell types, Ki values for BAU and BBAU did not vary regardless of the substrate used; that the values of Ki are different for the erythrocytes and L5178Y cells; and that BBAU is at least 5-fold more potent than BAU as an inhibitor of nucleoside transport. The inhibitory effects on the efflux of preloaded uridine indicate that BAU and BBAU are inhibitors, rather than permeants, of the nucleoside transport system.     

Cellular phosphorylation of 1-beta-D-arabinofuranosylcytosine 5-azacytidine with intact fibrosarcoma and leukemic cells.

The phosphorylation of 1-beta-D-arabinofuranosylcytosine (ara-C) and 5-azacytidine (5-aza-C) by A(T1)C1-3 hamster fibrosarcoma cells and L5178Y murine leukemic cells was studied, using intact cells. The cellular phosphorylation of both these nucleoside analogs appears to follow Michaelis-Menton kinetics. The apparent Km value for ara-C in the fibrosarcoma and leukemic cells was about 40 muM, whereas the apparent Km values for 5-aza-C in these cells were about 1.3 and 0.41 mM, respectively. Deoxycytidine and cytidine were found to be potent competitive inhibitors of the phosphorylation of ara-C and 5-aza-C, respectively, ara-C and 5-aza-C were found to be weak competitive inhibitors of the phosphorylation of deoxycytidine and cytidine, respectively. A clone isolated from the fibrosarcoma cells that was partially resistant to the cytotoxic effects of ara-C exhibited a higher Km value for both ara-C and deoxycytidine than the wild-type fibrosarcoma cells.