Amino acid-conferred protection against melphalan—Characterization of melphalan transport and correlation of uptake with cytotoxicity in cultured L1210 murine leukemia cells

The uptake of melphalan by L1210 cells was reduced to one-sixth of controls by physiological concentrations of the l-isomers of leucine and glutamine, and this decrease was accompanied by a corresponding decrease in cytotoxicity. Cytotoxicity was estimated by treatment of cells with melphalan for 35 min in phosphate-buffered saline containing bovine serum albumin and glucose followed by clonal growth of the surviving cells in soft agar. It was prominent within a critical region of net melphalan uptake of 2–5 pmoles/10⁵ cells. Inhibition analysis revealed that at cytotoxic concentrations melphalan is transported by a high-affinity amino acid transport system of the leucine (l) type. The values of the Michaelis constants (K_m) for l-leucine, a protective amino acid, l-valine, a minimally protective amino acid, and melphalan were 6, 58 and 19 μM respectively. These results suggest that the ability of amino acids to protect L1210 cells from melphalan cytotoxicity is related to their affinities for the leucine carrier sites.     

Aromatic l-amino acid decarboxylase activity in central and peripheral tissues and serum of rats with l-dopa and l-5-hydroxytryptophan as substrates

This paper describes the distribution of aromatic l-amino acid decarboxylase (AADC) activities in seventeen tissues (eleven peripheral and six brain tissues) of rats, using both l-DOPA and l-5-hydroxytryptophan (l-5-HTP) as substrates. The ratios of the activities of the enzyme using both substrates in the same homogenates were also determined. Rat pineal gland had the highest activity with the substrates followed by the liver, kidney and adrenals. Activity in the adrenals was not only high, but the ratio of the activities for l-DOPA and l-5-HTP was the highest. AADC activity was detected in rat serum by a new and highly sensitive high-performance liquid chromatography-voltammetric assay, using l-DOPA and l-5-HTP as substrates; the ratio of the activities for the two substrates was the lowest. K_m values, measured for the two substrates using the homogenates of the pineal, liver, and adrenals of rats, were found to be similar, but K_m values for l-DOPA were about 3-fold higher than those for l-5-HTP in the three tissues.     

The effect of alkylating agents and other drugs on the accumulation of melphalan by murine L1210 leukaemia cells in vitro

The effect of cytotoxic and other drugs on the accumulation of melphalan by L1210 murine leukaemia cells was studied. We have confirmed that uptake is an active process competitively inhibited by l-leucine. In 36 experiments in amino acid-free medium the mean concentration of melphalan taken up was 225 pmoles/10⁶ cells. High pressure liquid Chromatographie analysis showed that the majority of the drug is present as free native melphalan. 1, 3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) was the only drug that stimulated accumulation, but without significant effect on influx or efflux rates. Busulphan, chlorambucil, cyclophosphamide, interferon, methotrexate and prednisolone had no effect on accumulation after 30 min melphalan transport. Adriamycin, CCNU, methyl CCNU, mustine and vincristine all impaired melphalan accumulation as did the non-cytotoxic drugs aminophylline, chlorpromazine and ouabain. Adriamycin, aminophylline, chloropromazine, indomethacin and ouabain all reduced melphalan influx.     

Studies on the mechanism of resistance of selected murine tumors to l-alanosine

Sublines of P388 and L1210 leukemia were rendered resistant to l-alanosine [l-2-amino-3-(N-hydroxy-N-nitrosamino) propionic acid] and designated P388/LAL and L1210/LAL. Assessments were made of certain biochemical and pharmacological determinants of the sensitivity or resistance to l-alanosine of these sensitive and resistant lines. It was observed that the antibiotic strongly inhibited adenylosuccinate synthetase and DNA synthesis only in the parent or sensitive lines; moreover, after a therapeutic dose of the drug, the concentration of l-alanosyl-AICOR (l-alanosyl-5-amino-4-imidazole carboxylic acid ribonucleotide), the putative active anabolite of l-alanosine, was dramatically higher in these parent lines as compared with the resistant variants. Enzymologic studies established that, in P388/LAL, the specific activity of the enzyme SAICAR synthetase (5-amino-4-imidazole-N-succinocarboxamide ribonucleotide synthetase), which is believed to conjugate l-alanosine with the nascent purine AICOR (5-amino-4-imidazole carboxylic acid ribonucleotide), was depressed significantly; the same was not true for L1210/LAL. In both resistant lines, however, the enzymes of purine salvage were present at levels about 200 per cent higher than those measured in the native strains. These studies establish that resistance to l-alanosine is very likely pluricausal, but that the ability of susceptible cells to synthesize or retain l-alanosyl-AICOR is an element important to the process.     

Interaction of l-alanosine (NSC 153, 353) with enzymes metabolizing l-aspartic acid, l-glutamic acid and their amides

A comprehensive analysis has been made of the manner in which l-alanosine [l-2-amino-3-(N-nitroso, N-hydroxy) aminopropionic acid] interacts with the enzymes responsible for the metabolism of the dicarboxylic amino acids and their amides. The drug impedes the transport of l-aspartic acid and, to a lesser degree, of l-glutamic acid, l-asparagine and l-glutamine by lymphoblasts. in vitro; in each of these instances, inhibition is apparently competitive in type. Of the enzymes involved in the metabolism of l-aspartic acid, adenylosuccinate synthetase (EC 6.3.4.4). SAICAR synthetase (5-amino-4-imidazole-N-succinocarboxamide ribonucleotide synthetase) (EC 6.3.2.6). l-aspartyl tRNA synthetase (EC 6.1.1.12), l-aspartate transcarbamylase (EC 2.1.3.2) and l-aspartate aminotransferase (EC 2.6.1.1) were inhibited by l-alanosine; moreover, each of these enzymes except l-aspartyl tRNA synthetase accepted the antibiotic as substrate, although at substantially diminished rates. Of the enzymes involved in the metabolism of l-glutamic acid, l-alanosine inhibited only l-glutamine synthetase (EC 6.3.1.2) and l-glutamate decarboxylase (EC 4.1.1.15) to a prominent degree; this last enzyme was also capable of decarboxylating l-alanosine. Of the enzymes metabolizing l-asparagine and/or l-glutamine. only the l-glutaminase activity of l-asparagine amidohydrolase (EC 3.5.1.1) (with l-glutamine as substrate) and, to a lesser degree, carbamyl phosphate synthetase II (EC 2.7.2.9) were inhibited by the antibiotic. Although l-alanosine provokes a rise in the concentration of inosinic acid (IMP) in vitro, pointing to the conclusion that the drug is capable of inhibiting adenylosuccinate synthetase under these circumstances, no such rise was seen in vivo either in tumor or liver. However, 1 and 5 hr after administration l-alanosine depressed hepatic ATP and NAD pools, an effect which indicates that the drug is. in fact, restricting the intracellular concentration of adenine nucleotides. Of the metabolites of l-alanosine formed in vitro, α-decarboxy alanosine, α-keto alanosine, a-hydroxy alanosine. alanosyl IMP and N-carbamyll-alanosine did not inhibit adenylosuccinate synthetase to any prominent degree, whereas the metabolite generated by SAICAR synthetase powerfully inhibited this enzyme, with a K_i, of 0.3μM. Parenteral therapeutic doses of l-alanosine produced striking increases in the concentrations of l-aspartic acid in tumor and liver as well as of l-aspartic and l-glutamic acids in urine. It is concluded that the N-hydioxy, N-nitroso functionality of l-alanosine is analogous in structure to the β-carboxyls of l-aspartic and l-glutamic acids, respectively; this analogy permits l-alanosine to be anabolized and catabolized via several of the enzymatic routes which ordinarily operate on these dicarboxylic amino acids.     

Effects of a membrane sugar analogue, 6-deoxy-6-fluoro-d-galactose,on the L1210 leukemic cell ectosialyltransferase system

In L1210 leukemia cells, 6-deoxy-6-fluoro-d-galactose specifically inhibited the incorporation of [³H]-d-galactose, while that of other precursors of glycoconjugate biosynthesis, including mannose and glucosamine, was unaffected. The activation of [6-³H]-6-deoxy-6-fluoro-d-galactose to a nucleotide sugar was similar to that found for [³H]-d-galactose. The incorporation of either sugar after 1 hr was visualized by electron microscopic autoradiography to be in the Golgi region. Treatment of L1210 cells with 6-deoxy-6-fluoro-d-galactose in vitro or in vivo resulted in a specific, dose- and time-dependent decrease in the activity of cell surface sialyltransferase (ectosialyltransferase) but not of 5′-nucleotidase, a plasma membrane marker enzyme. The decrease in ectosialyltransferase activity appeared to be selective and is suggested to be due to structural modification of the cell surface galactoprotein acceptors for this enzyme. The data indicate that 6-deoxy-6-fluoro-d-galactose is an effective modifier of cellular glycoconjugate in that its incorporation into certain cell surface components results in a modification of plasma membrane structure and function.     

A fluorescent analogue of methotrexate as a probe for folate antagonist molecular receptors

A dansyl-l-lysine analogue of methotrexate, $\text{N}{}^{\mathrm{\alpha }}\text{-(}\text{4-amino-4-deoxy-10-methylpteroyl}\text{)-N}{}^{\mathrm{\epsilon }}\text{-(5-[N,N-}\text{dimethylamino]-1-naphthalenesulfonyl]-1-naphthalenesulfonyl)-}\text{l}\text{-lysine}$, is a potent inhibitor of murine L1210 dihydrofolate reductase. The dansyl fluorescence emission was enhanced approximately 3-fold with a 10 nm blue shift upon binding to L1210 dihydrofolate reductase. The fluorescent analogue was only 10-fold less potent than methotrexate in inhibiting the growth of methotrexate-sensitive and -resistant L1210 cells and competes effectively for [³H]methotrexate transport with a K_i of 7.02 μM, a value virtually identical to the K_t for methotrexate in both cell lines. In addition, strong dansyl fluorescence was found to be associated with dihydrofolate reductase from methotrexate-resistant, dihydrofolate reductase-overproducing L1210 cells following incubation of viable cells with the fluorescent methotrexate analogue for 4 hr. The results demonstrate that the dansyl-l-lysine analogue of methotrexate was rapidly transported into L1210 cells where it formed a high-affinity, fluorescent complex with intracellular dihydrofolate reductase.     

Mechanism of cytotoxic action of perfluorinated acids: II. Disruption of mitochondrial bioenergetics

PFAs and derivatives due to perfect technological properties are broadly applied in industry and consumer goods, and in consequence widely disseminated, environmentally bioaccumulative and found at ppb level in human serum.Earlier we revealed that in vitro cytotoxicity increases with chain length (CF₆-CF₁₄). The compounds dissipate plasma membrane potential and acidify of cytosol. Here we determine whether there is an association between the protonophoric uncoupling of respiration and disruption of bioenergetics caused by CF₆-CF₁₂ on HCT116 cell apoptosis.Again the effects were stronger for longer molecules. Incubation of cells with CF₁₀ stimulated time-dependent generation of reactive oxygen species, opening of mitochondrial permeability transition (MPT) pore, release of cytochrome c, activation of caspases and depletion of intracellular level of ATP occurring in intrinsic pathway of apoptosis. Incubation with decanoic acid (DA) did not lead to mitochondrial dysfunctions neither to cell cycle disturbances. Synchronized removal of the phosphorylated state of Akt, ERK1/2 and PKCδ/θ kinases by CF₁₀ suggests presence of concerted action to uninhibit Bad protein activation and a cascade of intrinsic pathway of apoptosis. Blocking MPT pore by cyclosporin A (CsA) led to a reduction of mitochondrial potential dissipation (mtΔΨ). Such cells neither showed cytochrome c release nor the downstream activation of caspase-9 and caspase-3. Our results confirm that mitochondria play a crucial role in perfluorochemicals induced apoptosis by releasing apoptotic signals through MPT pore.     

Analog specific aberrancies in antifolate inhibition of L1210 cell dihydrofolate reductase

During studies with L1210 cells and a variety of folate analogs, large discrepancies were revealed between data on membrane transport, on inhibition of dihydrofolate reductase in cell-free extracts, and on inhibition of growth in culture for 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin, and for 3-deaza, 10-methyl-aminopterin. While aminopterin, 10-methyl (methotrexate)-, 10-ethyl- and 10-propyl-aminopterin were tight binding inhibitors (K_i: 2–3 × 10^?12M) of dihydrofolate reductase in cell-free extracts from L1210 cells, the other four analogs were only weak competitive inhibitors (K_i = 3–300 × 10^?8M). Similar differences among analogs were observed for inhibition of dihydrofolate reductase in cell-free extracts from Sarcoma 180 and Ehrlich cells, but not for this enzyme in microbial cell-free extracts. There were only small differences in the transport of all of the analogs by L1210 cells. Inhibition of L1210 cell growth in culture by 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin and by 3-deaza, 10-methyl-aminopterin, in contrast to the other analogs, was several orders of magnitude greater than that predicted from the data on dihydrofolate reductase inhibition. The extent of binding of 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin, and of 3-deaza and 10-methyl-aminopterin to dihydrofolate reductase in intact L1210 cells, in contradistinction to that seen for the cell-free enzyme preparations, approached that observed for methotrexate; these estimates of drug-enzyme interaction in situ were more predictive of the extent of inhibition by these analogs of L1210 cell growth in culture.     

Effects of bis(6-mercaptopurine-9-beta-D-ribofuranoside)-5',5"-phosphate and its butyryl derivative on mouse leukaemia L1210 and a 6-mercaptopurine-resistant subline in culture

Bis(6-mercaptppurine-9-β-d-ribofuranoside)-5′-5″′-monophosphate (bis(MPR)P) and its butyryl derivative, bis(O²,$\text{O}{}^3\text{-}\text{dibutyryl}\text{-6-}\text{mercaptopurine}\text{-9-β-}\text{d}\text{-}\text{ribofuranoside}$)-5′,5″′-monophosphate (bis(dibutyrylMPR)P) were synthesized from 6-mercaptopurine-9-β-d-ribofuranoside (MPR). Bis(MPR)P (ec₅₀ = 0.014 μM) and MPR (ec₅₀ = 0.022 μM) were essentially equivalent in their growth inhibitory activities against L1210/0 cell cultures, whilst bis(dibutyrylMPR)P (ec₅₀ = 1.1 μM) was considerably less effective. L1210/MPR cells grew normally in the presence of 1 mM MPR but were inhibited by bis(MPR)P (ec₅₀ = 580 μM) and (bis(dibutyrylMPR)P (ec₅₀ = 42 μM). Bis(dibutyrylMPR)P was less readily broken down to MPR by enzymes in the serum component of the culture medium than was bis(MPR)P, and leukaemia cells did not contribute to the extracellular degradation of the acylated derivative. The delayed cytotoxic effects of bis(MPR)P and bis(dibutyrylMPR)P on L1210/0 cells were those of the MPR breakdown product. Exposure to bis(MPR)P resulted in delayed cytotoxicity in L1210/MPR cultures whilst bis(dibutyrylMPR)P produced only acute growth inhibition and no delayed effect on the MPR-resistant subline. MPR was incorporated into DNA of L1210/0 cells as 6-thioguanine deoxyribonucleotide whilst bis(MPR)P was not incorporated into L1210/MPR cell DNA. These results suggested that the ultimate mechanisms of action of bis(MPR)P and bis(dibutyrylMPR)P in L1210/ MPR cells may have been different from that of MPR in sensitive L1210/0 cells and therefore might not represent true circumvention of resistance to MPR.     

N-Methylformycins. Reactivity with adenosine deaminase, incorporation into intracellular nucleotides of human erythrocytes and L 1210 cells and cytotoxicity to L 1210 cells.

The N-methyl derivatives of the C-nucleoside, formycin (7-amino-3(β-d-ribofurano-syl)pyrazolo[4, 3-d]pyrirnidine) were compared to formycin and adenosine with regard to their substrate activity with human erythrocytie adenosine deaminase (ADA), their ability to form intracellular nucleotides and their cytotoxicity to L1210 cells. Only 2-methylformycin (K_m = 6.1 mM, relative V_max = 396) and N^? -methylformycin (K_m = 0.1 mM, relative V_max = 3) showed substrate activity with ADA (corresponding kinetic parameters for adenosine were: K_m = 0.025 mM, relative V_max = 100). In contrast to previous hypotheses, these results suggest that the conformation (either syn or anti) of an adenosine analog is not a major factor in determining substrate activity with ADA. Neither 4-methylformycin nor 6-methylformycin formed their corresponding nucleotides when incubated with human erythrocytes, whereas both 1-methylfor-mycin and 2-methylformycin formed large amounts of their corresponding mono-, di- and triphosphate nucleotides. Inhibition of ADA by pretreatment of the erythrocytes with the potent ADA inhibitor, 2'-deoxycoformycin, had no effect on the incorporation of 1-methylformycin into erythrocytic nucleotides but greatly increased the incorporation of 2-methylformycin and N⁷-methylformycin. The conversion of both 1-methylformycin and 2-methylformycin into nucleotides was almost complete after 18 hr of incubation (in the presence of 2'-deoxycoformycin in the case of 2 methylformycin), whereas that of N⁷-methylformycin was only partially complete in the presence of 2'-deoxycoformycin. With both 1-methylformycin and N⁷-methylformycin, transient accumulation of the corresponding nucleoside 5'-monophosphate derivative was observed prior to the accumulation of the triphosphate nucleotide. Results, qualitatively similar to those found with erythrocytes, were obtained when the effects of 2'-deoxycoformycin on the incorporation of 1-methyl- and 2-methylformycins into the nucleotide pools of L 1210 cells in vitro were examined. Compounds capable of forming analog nucleotides in human erythrocytes or L1210 cells if deamination is prevented either by the molecular structure of the analog or by pretreatment of the cells with 2'-deoxycoformycin, also showed marked cytotoxicity to L1210 cells in culture, i.e. 1-methyl-, 2-methyl- and N⁷-methylformycin exhibited id₅₀ values of 0.5 to 2 μM, whereas 4-methyl- and 6-methylformyein were not significantly growth inhibitory. The potential usefulness of the various N-methyl derivatives of formycin (alone or in combination with an ADA inhibitor) as cytotoxic or antiviral agents is discussed.     

Further studies of stereospecificity at carbon 6 for membrane transport of tetrahydrofolates: Diastereoisomers of 5-methyltetrahydrofolates as competitive inhibitors of transport of methotrexate in L1210 cells

The unnatural d diastereoisomer at carbon 6 of 5-methyltetrahydrofolate was only slightly less effective than the natural l diastereoisomer as a competitive inhibitor of the carrier-mediated membrane transport of [³H]methotrexate into L1210 murine leukemia cells. The apparent K_i for a mixture containing equal amounts of both natural and unnatural diastereoisomers was not significantly different from that found for the unnatural form. These results show that the reduced folate carrier system in these cells has a strong affinity for the unnatural stereoisomer, a finding in contrast to that obtained with the corresponding diastereoisomer of 5-formyltetrahydrofolate.     

Inhibitors of purine nucleoside phosphorylase, C(8) and C(5') substitutions

The C(8) and C(5') positions of base and nucleoside substrates of human erythrocytic purine nucleoside phosphorylase (PNP) are promising sites for the development of an inhibitor of this enzyme. The substrate analog, 8-aminoguanine (8-AG), has the lowest dissociation constant (K_i = 0.2–1.2 μM) of any compound reported to date and V_max = 16 per cent (relative to guanine). Other C(8) substituents decreased the affinity for PNP and, with the exception of the methyl and sulfhydryl groups, abolished substrate activity. Halogens or a thiomethyl group at C(5') of inosine resulted in unchanged or improved affinities (K_i = 10–30 μM) and greatly decreased substrate activity (V_max < 1 per cent relative to inosine). The K_i of formycin B was reduced from 100 μM to 10 μM or less by substitution of a halogen at C(5'). Phosphorolysis of purine nucleosides was inhibited significantly by 8-AG in intact human erythrocytes and murine Sarcoma 180, L1210 and L5178Y cells. Although a K_i value of 17 μM was determined for 8-aminoguanosine, it was equally effective in inhibiting PNP activity in intact cells. The nucleoside was cleaved to 8-AG which was not a substrate for guanase or hypoxanthine-guanine phosphoribosyltransferase. Despite its low substrate activity (V_max < 0.2%). 5′-deoxy-5′-iodoinosine was cleaved by intact L1210 and L5178Y cells.     

Stereospecificity at carbon 6 of fomyltetrahydrofolate as a competitive inhibitor of transport and cytotoxicity of methotrexate in vitro

The unnatural diastereoisomer of l-5-formyltetrahydrofolate was 20-fold less effective as a competitive inhibitor of [³H] methotrexate influx than the natural diastereoisomer during carrier-mediated membrane transport in L1210, S180 and Ehrlich cells. Values derived for K_i, were 1.84 to 2.29 μM for the natural derivative and 35.2 to 53.8 μM for the unnatural derivative. Values for K_i derived with a chemically synthesized mixture containing equal amounts of both natural and unnatural diastereoisomers were 2-fold greater than values obtained for the natural diastereoisomer. The unnatural diastereoisomer was 100-fold less effective and the chemically synthesized mixture was 2-fold less effective than the natural diastereoisomer in preventing inhibition by methotrexate of L1210 cell growth in culture. These results indicate that the unnatural diastereoisomer competes relatively ineffectively with the natural diastereoisomer or methotrexate for transport in these murine tumor cells.     

The signalling role of action potential depolarization in insulin secretion: Metabolism-dependent dissociation between action potential increase and secretion increase by TEA

The K⁺ channel blocker, TEA is known to increase action potential amplitude and insulin secretion of mouse β-cells when added to a nutrient secretagogue. In the presence of a maximally effective sulfonylurea concentration (2.7 μM glipizide) the nutrient secretagogue α-ketoisocaproic acid (KIC, 10 mM) strongly increased insulin secretion (about elevenfold). Instead of enhancing the effect of KIC, TEA reduced the KIC-induced secretion by more than 50%. Also, the secretion rate produced by 2.7 μM glipizide alone was significantly reduced by TEA. In contrast, TEA enhanced the insulinotropic effect of glipizide when a basal glucose concentration (5 mM) was present. In the presence as well as in the absence of glucose glipizide produced a plateau depolarization with superimposed action potentials. Under both conditions, TEA increased the glipizide-induced action potential amplitude and further elevated the cytosolic free calcium concentration ([Ca²⁺]_i) with an oscillatory characteristic. These effects depended on the activity of L-type Ca²⁺ channels, even though the effect of TEA differed from that of 1 μM of the Ca²⁺ channel opener, Bay K8644, which primarily increased action potential duration. TEA did not negatively affect parameters of β-cell energy metabolism (NAD(P)H fluorescence and ATP/ADP ratio), rather, it slightly increased NAD(P)H fluorescence. Apparently, TEA inhibits insulin secretion in the absence of glucose in spite of a persistent ability to block K⁺ ion conductance. Thus, the signalling role of action potential depolarization in insulin secretion may require reconsideration and ion conductance-independent actions of K⁺ channels may be involved in this paradox effect of TEA.     

Studies on rat kidney 15-hydroxy-prostaglandin dehydrogenase.

Rat kidney 15-hydroxy-prostaglandin dehydrogenase (PGDH) was isolated, and its characteristics and the effects of various drugs upon it were examined. The enzyme was found in the cell cytosol; was labile when unfrozen; and most active at alkaline pH, at 41°, and with the E prostaglandins. Additionally, the enzyme was inhibited by furosemide (K_i = 0.019 mM), ethacrynic acid (K_i = 0.27 mM, phenylbutazone (K_i = 0.16 mM), acetylsalicylic acid (K_i = 3.8 mM), and potassium cyanide (K_i = 1.03 mM). Inhibition of PGDH may play a role in the mechanism of action of the diuretic and anti-inflammatory drugs. Little or no inhibition was seen with amobarbital, hydralazine, alpha-methyldopa, bethanidine and guanethidine. Amobarbital inhibits NADH oxidase (K_i = 0.5 mM), but does not inhibit PGDH. This drug, therefore, may be useful in permitting the use of the fluorometric assay for PGDH in preparations of PGDH contaminated by NADH oxidase.     

Binding of copper and zinc by the antitumour agent l-alanosine

l-Alanosine [l-2-amino-3(hydroxynitrosamino)proprionic acid], an anticancer agent recently introduced into clinical trial, formed a 1:1 complex with Cu(II) with an effective stability constant (log K_eff at pH 7.2, determined by the method of competing equilibria, of 22.7 and with Zn(II) of 17.2. The intravenous administration of l-alanosine to rabbits at a dose of 30 mg/kg resulted in an immediate decrease in total plasma copper of up to 17 per cent. Plasma copper levels returned to normal within 24 hr. There was no increase in urinary copper excretion and a 4-fold increase in urinary zinc excretion. Cancer patients receiving l-alanosine also had a transient decrease in plasma copper (up to 30 per cent) and an increase in urinary zinc excretion. There was no significant difference in the antitumor effects of l-alanosine against intraperitoneally transplated P-388 leukemia in normal, copper-deficient, or copper-loaded mice. The Cu(II) complex of l-alanosine lacked antitumor activity at doses that were tolerated by the mice. The increased toxicity of the Cu(II) complex of l-alanosine compared to l-alanosine was attributable to the Cu(II).     

Effects of benserazide and carbidopa on the metabolism of l-tryptophan by isolated rat liver cells

(1) The effects of two inhibitors of aromatic amino acid decarboxylase, benserazide and carbidopa, on the metabolism of l-tryptophan by liver cells prepared from 48 hr starved rats was studied. Tryptophan 2,3-dioxygenase (pyrrolase) and kynureninase activities were determined simultaneously using l-[ring 2-¹⁴C], l-[carboxyl-¹⁴C] and l-[methylene-¹⁴C]tryptophan. (2) In hepatocytes incubated with a low physiological concentration (0.1 mM) of tryptophan, carbidopa (5 × 10^?5M) produced a significant inhibiton of kynureninase activity. Inhibition of tryptophan dioxygenase flux occurred only at concentrations of carbidopa of 10^?4 M and above. Benserazide was less potent; the concentrations required for significant inhibition of tryptophan dioxygenase and kynureninase fluxes were 2.5 × 10^?4 M and 10^?4 M respectively. (3) Rates of ¹⁴CO₂ production from the two side chain ¹⁴C-labelled tryptophan radioisomers were inhibited by benserazide and carbidopa to much greater extents than were rates of [1-¹⁴C]alanine and [3-¹⁴C]alanine release. This phenomenon is consistent with the finding that both drugs block oxidation of alanine by inhibiting transamination. Neither compound influences pyruvate oxidation. The implications of these observations with reference to the interpretation of data from previous investigations of the effect of benserazide and carbidopa on tryptophan oxidation in vivo are discussed.     

Studies with a 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (DDMP)-resistant L1210 leukemia cell line without cross-resistance to methotrexate

An L1210 leukemia cell line resistant to 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (DDMP) (L1210/DDMP) was developed in vivo by treatment of tumor-bearing mice. Resistance to DDMP was confirmed by subsequent in vivo survival experiments and by in vitro dose-response curves. The L1210/DDMP line demonstrated little cross-resistance to another folate analog, methotrexate (MTX). This was confirmed both in vivo, with survival experiments, and in vitro, using dose-response curves. A statistical analysis of the in vivo data confirmed DDMP resistance with lack of MTX cross-resistance. Dihydrofolate reductase (DHFR) activity in the L1210/DDMP/R₅ line was no greater than in the parent cell line (L1210/S). and the K_m of DHFR for dihydrofolate was the same in the L1210/DDMP/R₅ and L1210/S lines. The K_i for DHFR of the L1210/DDMP/R₅ cell line versus the L1210/S cell line was increased 3.0-fold for MTX and 3.5-fold for DDMP. Total accumulation of [¹⁴C]DDMP was identical in the two cell lines. The explanation for the lack of MTX cross-resistance in the L1210/DDMP/R₅ line is unknown.     

Perchlorate transport and inhibition of the sodium iodide symporter measured with the yellow fluorescent protein variant YFP-H148Q/I152L

Perchlorate is an environmental contaminant that impairs thyroid function by interacting with the sodium iodide symporter (NIS), the transporter responsible for iodide uptake in the thyroid gland. Perchlorate is well known as a competitive inhibitor of iodide transport by NIS, and recent evidence demonstrates that NIS can also transport perchlorate. In this study, we evaluated the yellow fluorescent protein (YFP) variant YFP-H148Q/I152L, as a genetically encodable biosensor of intracellular perchlorate concentration monitored by real-time fluorescence microscopy. Fluorescence of recombinant YFP-H148Q/I152L was suppressed by perchlorate and iodide with similar affinities of 1.2 mM and 1.6 mM, respectively. Perchlorate suppressed YFP-H148Q/I152L fluorescence in FRTL-5 thyroid cells and NIS-expressing COS-7 cells, but had no effect on COS-7 cells lacking NIS. Fluorescence changes in FRTL-5 cells were Na⁺-dependent, consistent with the Na⁺-dependence of NIS activity. Perchlorate uptake in FRTL-5 cells resulted in 10-fold lower intracellular concentrations than iodide uptake, and was characterized by a higher affinity (K_m 4.6 μM for perchlorate and 34.8 μM for iodide) and lower maximal velocity (V_max 6.8 μM/s for perchlorate and 39.5 μM/s for iodide). Perchlorate also prevented iodide-induced changes in YFP-H148Q/I152L fluorescence in FRTL-5 cells, with half-maximal inhibition occurring at 1.1-1.6 μM. In conclusion, YFP-H148Q/I152L detects perchlorate accumulation by thyroid and other NIS-expressing cells, and reveals differences in the kinetics of perchlorate versus iodide transport by NIS.