thioinosine and Lymphoma

The poor absorption of orally administered 6-mercaptopurine (6MP) causes a wide variation in its cytotoxic efficacy. An i.v. dosage form would eliminate this problem. Our objective was to compare the pharmacokinetics of 6MP administered orally with those of an i.v. dosage form 6-mercaptopurine riboside (6MPR), in children with acute lymphoblastic leukemia or malignant lymphoma.. A total of 10 children were treated with oral 6MP, 50 mg/m(2) per day, while five children were treated with 6MPR, 50 mg/m(2) per day, administered by rapid i.v. injection. The plasma concentrations of 6MP and of 6MPR were measured on day 0, while the concentrations of 6-thioguanine nucleotides (6TGN) in red blood cells (RBC) were measured on day 2. The area under the plasma concentration-time curve (AUC1-5) was calculated from 1 to 5 h after drug administration.. With the intravenously administered 6MPR, the AUC1-5 ranged from 124 to 186 (1.5-fold range, median 145) microM min; only two samples were obtained for the RBC concentration of 6TGN, and were 121 and 273 pmol per 25 mg hemoglobin. With the orally administered 6MP, the AUC1-5 ranged from 23 to 65 microM min (2.8-fold range, median 56); the RBC concentration of 6TGN ranged from 18 to 152 pmol per 25 mg hemoglobin (median 75).. The i.v. administration of 6MPR showed less interindividual variation in the AUC1-5 coupled with a higher RBC level of 6TGN as compared with those by oral 6MP. We conclude that the i.v. administration of 6MPR achieves stable blood levels of active drug in children undergoing cancer chemotherapy.

Topics: Administration, Oral; Adolescent; Antimetabolites, Antineoplastic; Area Under Curve; Child; Child, Preschool; Female; Humans; Injections, Intravenous; Lymphoma; Male; Mercaptopurine; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Thioinosine

Topics: Biological Transport; Carrier Proteins; Humans; Leukemia; Lymphocytes; Lymphoma; Membrane Proteins; Nucleoside Transport Proteins; Pentostatin; Thioinosine; Tumor Cells, Cultured

S49 murine lymphoma cells were examined for expression of various nucleoside transport systems using a non-metabolized nucleoside, formycin B, as substrate. Nitrobenzylthioinosine (NBTI)-sensitive, facilitated transport was the primary nucleoside transport system of the cells. The cells also expressed very low levels of NBTI-resistant, facilitated nucleoside transport as well as of Na(+)-dependent, concentrative formycin B transport. Concentrative transport was specific for uridine and purine nucleosides, just as the concentrative nucleoside transporters of other mouse and rat cells. A nucleoside transport mutant of S49 cells, AE-1, lacked both the NBTI-sensitive, facilitated and Na(+)-dependent, concentrative formycin B transport activity, but Na(+)-dependent, concentrative transport of alpha-aminoisobutyrate was not affected.

Topics: Aminoisobutyric Acids; Animals; Biological Transport, Active; Carrier Proteins; Dipyridamole; Formycins; Kinetics; Lymphoma; Membrane Proteins; Mice; Mutation; Nucleoside Transport Proteins; Nucleosides; Sodium; Thioinosine; Tumor Cells, Cultured

A novel type of somatic mutation that causes the expression of a high affinity purine base permease has been inserted into murine S49 lymphoma cells that are deficient in nucleoside transport. Two classes of mutants expressing this nucleobase permease were generated. The first, as exemplified by the AE1HADPAB2 cell line, possesses an augmented capacity to transport low concentrations of the three purine bases, hypoxanthine, guanine, and adenine. The second class of mutants, as typified by the AE1HADPAB5 clone, possesses an augmented capability to translocate low levels of hypoxanthine and guanine, but not adenine. Neither the AE1HADPAB2 nor the AE1HADPAB5 cells can transport nucleosides suggesting that the expression of the high affinity base transporter did not revert the mutation in the nucleoside transport system. Fusion of the AE1HADPAB2 and AE1HADPAB5 cell lines with wild type cells indicated that the expression of the high affinity base transporter behaved in a dominant fashion, while the nucleoside transport deficiency was a recessive trait. These data suggest that the high affinity purine base transporter of mutant cells and the nucleoside transport function of wild type cells are products of different genes and that expression of the former probably requires the unmasking or alteration of a specific genetic locus that is silent or different in wild type cells.

Topics: Adenine; Animals; Biological Transport; Clone Cells; Dipyridamole; Dose-Response Relationship, Drug; Gene Expression; Guanine; Hybridization, Genetic; Hypoxanthine; Hypoxanthines; Inosine; Lymphoma; Mice; Mice, Inbred BALB C; Mutation; Phenotype; T-Lymphocytes; Thioinosine; Tumor Cells, Cultured

Dilazep, a tertiary amine that is greater than 96% protonated at pH 7.4, is a potent inhibitor of facilitated diffusion (equilibrative) nucleoside transport (NT) in animal cells. In this study, saturable reversible binding of [3H]dilazep was demonstrated at sites on S49 mouse lymphoma cells but not in AE1 cells, an NT-deficient mutant of S49 cells. Mass law analysis of dilazep binding under equilibrium conditions revealed two saturable components, representing binding sites that differed about 50-fold in affinity for dilazep (Kd values of 0.21 and 10 nM). At pH 7.4, the low affinity sites were more abundant (Bmax, 3.5 X 10(5) sites/cell) than the high affinity site (Bmax, 3.0 X 10(4) sites/cell). Binding of dilazep was pH dependent; at pH 9.0, binding at the high affinity sites predominated, whereas, at pH 5.0, the low affinity component predominated, suggesting that these components represented binding of nonprotonated and protonated dilazep molecules, respectively. Nitrobenzylthioinosine (NBMPR) and physostigmine selectively blocked binding of nonprotonated and protonated species of dilazep, respectively, at pH 7.4, yielding Scatchard plots that were similar to control plots obtained at pH 5.0 and 9.0. First-order plots of the dissociation of [3H]dilazep-binding site complexes in the presence of excess nonradioactive dilazep at pH 7.4 were nonlinear and were resolved into rapid (rate constant, 3.4-4.7 min-1) and slow (rate constant, 0.13-0.15 min-1) components. In the presence of site-saturating concentrations of NBMPR or high concentrations of nucleoside permeants, dissociation of site-bound [3H]dilazep was incomplete and only the slow component of dissociation was apparent (rate constant, 0.11-0.19 min-1). The combined presence of nonradioactive dilazep and NBMPR yielded time courses of [3H]dilazep-site dissociation equivalent to those obtained in the presence of nonradioactive dilazep alone. These results are consistent with a model in which protonated and nonprotonated species of dilazep bind at separate sites on S49 cells. The absence of both high and low affinity sites on AE1 cells suggests that, in S49 cells, both populations of sites are associated with NT polypeptides. The high affinity sites that bind nonprotonated species of dilazep appear to overlap with NBMPR binding sites on these cells.

Topics: Animals; Azepines; Binding Sites; Carrier Proteins; Dilazep; Hydrogen-Ion Concentration; Inosine; Kinetics; Lymphoma; Membrane Proteins; Mice; Nucleoside Transport Proteins; Physostigmine; Thioinosine; Tumor Cells, Cultured

The uptake of various nucleosides by S49 mouse T-lymphoma cells and that by a single-step nucleoside transport-defective mutant thereof (AE1) were compared. Residual nucleoside entry into AE1 cells occurred via two routes, nonmediated permeation and saturable, non-concentrative transport with broad substrate specificity and a Michaelis-Menten constant approximating that for thymidine transport in wild-type cells. However, in contrast to nucleoside transport in wild-type cells, residual nucleoside transport in AE1 cells was resistant to inhibition by nitrobenzylthioinosine. In its properties the latter resembled nitrobenzylthioinosine-resistant nucleoside transport observed in other types of mammalian cells. It amounted to less than 1% of the total nucleoside transport activity of wild-type S49 cells. The results indicate that nitrobenzylthioinosine-resistant and -sensitive nucleoside transports are genetically distinguishable. In wild-type cells, the salvage of thymidine, when present at concentrations higher than 1 to 10 microM, was limited by phosphorylation, because of the saturation of thymidine kinase. In AE1 cells, entry into the cells mainly limited thymidine salvage, but at high thymidine concentrations the combined entry via residual transport and nonmediated permeation was sufficiently rapid to support intracellular thymidine phosphorylation at rates comparable to those observed in wild-type cells.

Topics: Animals; Carrier Proteins; Cell Line; Drug Resistance; Inosine; Kinetics; Lymphoma; Membrane Proteins; Mice; Mutation; Nucleoside Transport Proteins; Nucleosides; T-Lymphocytes; Thioinosine; Thymidine; Thymine Nucleotides

From a mutagenized population of wildtype S49 T lymphoma cells, clones were generated that were resistant to the physiological effects of the potent inhibitor of nucleoside transport, 4-nitrobenzyl-6-thioinosine (NBMPR). NBMPR protected wildtype cells from the cytotoxic effects of a spectrum of nucleosides, whereas two mutant clones, KAB1 and KAB5, were still sensitive to nucleoside-mediated cytotoxicity in the presence of NBMPR. In addition, NBMPR prevented wildtype cells from surviving in hypoxanthine-amethopterin-thymidine containing medium, whereas KAB1 and KAB5 cells grew normally. Rapid sampling transport studies indicated that mutant cells, unlike wildtype parental cells, had acquired a substantial NBMPR-insensitive nucleoside transport component. Binding studies with [3H]NBMPR indicated that KAB5 cells were 70-75% deficient in the number of NBMPR binding sites, whereas KAB1 cells possessed a wildtype complement of NBMPR binding sites. The characterization of the KAB1 and KAB5 cell lines suggested that the NBMPR binding site in wildtype S49 cells is genetically distinguishable from the nucleoside carrier site.

Topics: Animals; Binding Sites; Blood Proteins; Cell Line; Inosine; Lymphoma; Membrane Proteins; Mice; Nucleoside Transport Proteins; Ribonucleosides; Thioinosine

The characteristics of nucleoside transport were examined in Walker 256 rat carcinosarcoma and S49 mouse lymphoma cells. In Walker 256 cells the initial rates of uridine, thymidine and adenosine uptake were insensitive to the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR) (1 microM), but were partially inhibited by dipyridamole (10 microM), another inhibitor of nucleoside transport. In contrast, the transport of these nucleosides in S49 cells was completely blocked by both inhibitors. Nucleoside transport in Walker 256 and S49 cells also differed in its sensitivity to the thiol reagent p-chloromercuribenzenesulphonate (pCMBS). Uridine transport in Walker 256 cells was inhibited by pCMBS with an IC50 (concentration producing 50% inhibition) of less than 25 microM, and inhibition was readily reversed by beta-mercaptoethanol. In S49 cells uridine transport was only inhibited at much higher concentrations of pCMBS (IC50 approximately equal to 300 microM). In other respects nucleoside transport in Walker 256 and S49 cells were quite similar. The Km and Vmax. values for uridine transport were nearly identical, and the transporters of both cell lines appeared to accept a broad range of nucleosides as substrates. Uridine transport in Walker 256 cells was non-concentrative and did not require an energy source. These studies demonstrate that nucleoside uptake in Walker 256 cells is mediated by a facilitated-diffusion mechanism which differs markedly from that of S49 cells in its sensitivity to the transport inhibitor NBMPR and the thiol reagent pCMBS.

Topics: 4-Chloromercuribenzenesulfonate; Animals; Biological Transport; Carcinoma 256, Walker; Cell Line; Dipyridamole; Inosine; Kinetics; Lymphoma; Mice; Nucleosides; Rats; Sulfhydryl Compounds; Thioinosine; Uridine

A mutant clone (NT-1) of a T-cell lymphoma was selected for its ability to grow in HAT medium (hypoxanthine, aminopterin and thymidine) in the presence of the nucleoside transport inhibitor P-nitrobenzyl-6-mercaptoinosine (NBMI). NT-1 cells contain half the number of NBMI binding sites present on the parental S49 cells and are partially able to transport nucleosides in the presence of the transport inhibitor (NBMI). These observations suggest that the mutant cells are heterozygous for nucleoside transport proteins and contain two types of transport proteins: the first protein can both bind and is inhibited by NBMI similar to the wild type phenotype, and the second is an altered protein. The altered transport protein apparently lost its NBMI binding sites without a parallel loss of nucleoside transport ability suggesting that the nucleoside transported sites are separate from the binding sites of the transport inhibitor.

Topics: Adenosine; Animals; Biological Transport; Cell Division; Cells, Cultured; Deoxycytidine; Kinetics; Lymphoma; Mice; Nucleosides; Thioinosine; Thymidine

From a mutagenized population of wild type S49 T lymphoma cells, clones were generated that were resistant to the physiological effects of the potent inhibitor of nucleoside transport, 4-nitrobenzyl-6-thioinosine (NBMPR). These cells were selected for their ability to survive in semisolid medium containing 0.5 mM hypoxanthine, 0.4 microM methotrexate, 30 microM thymidine, 30 microM deoxycytidine, in the presence of 30 microM NBMPR. NBMPR protected wild type cells from the effects of a spectrum of cytotoxic nucleosides, whereas two mutant clones, KAB1 and KAB5, were still sensitive to nucleoside-mediated cytotoxicity in the presence of NBMPR. Comparisons of the abilities of wild type cells and mutant cells to incorporate exogenous nucleoside to the corresponding nucleoside triphosphate indicated that the KAB1 and KAB5 mutant cells were refractory to normal inhibition by NBMPR. Moreover, rapid transport studies indicated that mutant cells, unlike wild type parental cells, had acquired a substantial NBMPR-insensitive nucleoside transport component. Binding studies with [3H]NBMPR indicated that KAB5 cells were 70-75% deficient in the number of NBMPR binding sites, whereas KAB1 cells possessed a wild type complement of NBMPR binding sites. These data suggest that the NBMPR binding site in wild type S49 cells is genetically distinguishable from the nucleoside carrier site.

Topics: Animals; Biological Transport, Active; Blood Proteins; Clone Cells; Hybrid Cells; Inosine; Lymphoma; Membrane Proteins; Mice; Mutation; Nucleoside Transport Proteins; Nucleosides; Thioinosine

The availability of a human lymphoma cell line deficient in adenosine deaminase, adenosine kinase and methylthioadenosine phosphorylase enabled us to compare the effects of nucleoside transport inhibitors on the excretion of endogenously generated adenosine, deoxyadenosine and 5'-methylthioadenosine. The nucleoside transport inhibitors nitrobenzylthioinosine and dipyridamole blocked the efflux of adenosine, but not deoxyadenosine or 5'-methylthioadenosine. The inhibitors also prevented the uptake of exogenous adenosine, but not deoxyadenosine or 5'-methylthioadenosine, by human lymphoblasts. The results show (i) that the transport inhibitors modify adenine nucleoside efflux and influx similarly, and (ii) that the effects of the compounds on the excretion and uptake of these three physiologically important adenine nucleosides are distinctly different.

Topics: Adenosine; Adenosine Deaminase; Adenosine Kinase; Biological Transport; Cell Division; Cell Line; Deoxyadenosines; Dipyridamole; Humans; Lymphocytes; Lymphoma; Nucleoside Deaminases; Purine-Nucleoside Phosphorylase; Thioinosine; Thionucleosides

Nitrobenzylthioniosine (NBMPR), a potent and specific inhibitor of nucleoside transport, is bound reversibly by high affinity sites on nucleoside transporter proteins of erythrocyte membranes and, upon photoactivation, NBMPR molecules become covalently bonded to the sites. This study showed that [3H]NBMPR molecules reversibly bound to intact S49 and L5178Y mouse lymphoma cells became covalently bound upon exposure to UV light. Electrophoretic analysis of plasma membrane fractions from the labelled cells showed that 3H was present in polypeptides which migrated as a major band with an apparent Mr of 45000-65000.

Topics: Affinity Labels; Animals; Blood Proteins; Cell Line; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Inosine; Leukemia L5178; Lymphoma; Membrane Proteins; Mice; Nucleoside Transport Proteins; Photochemistry; Thioinosine; Ultraviolet Rays

The zero-trans influx of 500 microM uridine by CHO, P388, L1210 and L929 cells was inhibited by nitrobenzylthioinosine ( NBTI ) in a biphasic manner; 60-70% of total uridine influx by CHO cells and about 90% of that in P388, L1210 and L929 cells was inhibited by nmolar concentrations of NBTI (ID50 = 3-10 nM) and is designated NBTI -sensitive transport. The residual transport activity, designated NBTI -resistant transport, was inhibited by NBTI only at concentrations above 1 microM (ID50 = 10-50 microM). S49 cells exhibited only NBTI -sensitive uridine transport, whereas Novikoff cells exhibited only NBTI -resistant uridine transport. In all instances NBTI -sensitive transport correlated with the presence of between 7 7 X 10(4) and 7 X 10(5) high-affinity NBTI binding sites/cell (Kd = 0.3-1 nM). Novikoff cells lacked such sites. The two types of nucleoside transport, NBTI -resistant and NBTI -sensitive, were indistinguishable in substrate affinity, temperature dependence, substrate specificity, inhibition by structurally unrelated substances, such as dipyridamole or papaverine, and inhibition by sulfhydryl reagents or hypoxanthine. We suggest, therefore, that a single nucleoside transporter can exist in an NBTI -sensitive and an NBTI -resistant form depending on its disposition in the plasma membrane. The sensitive form expresses a high-affinity NBTI binding site(s) which is probably made up of the substrate binding site plus a hydrophobic region which interacts with the lipophilic nitrobenzyl group of NBTI . The latter site seems to be unavailable in NBTI -resistant transporters. The proportion of NBTI -resistant and sensitive uridine transport was constant during proportion of NBTI -resistant and sensitive uridine transport was constant during progression of P388 cells through the cell cycle and independent of the growth stage of the cells in culture. There were additional differences in uridine transport between cell lines which, however, did not correlate with NBTI sensitivity and might be related to the species origin of the cells. Uridine transport in Novikoff cells was more sensitive to inhibition by dipyridamole and papaverine than that in all other cell lines tested, whereas uridine transport in CHO cells was the most sensitive to inactivation by sulfhydryl reagents.

Topics: Animals; Biological Transport; Cell Line; Cricetinae; Cricetulus; Female; Hypoxanthine; Hypoxanthines; Inosine; Kinetics; L Cells; Leukemia L1210; Leukemia P388; Liver Neoplasms, Experimental; Lymphoma; Mice; Ovary; Rats; Thioinosine; Tritium; Uridine

Plasma membrane-enriched fractions from disrupted S49 lymphoma cells contained high affinity sites for [3H]nitrobenzylthioinosine, a potent and specific inhibitor of nucleoside transport. These sites were absent from similar preparations from AE1 cells, a nucleoside-transport deficient clone derived from the S49 cell line. Reversible binding of [3H]nitrobenzylthioinosine to the S49 membrane preparations was inhibited by adenosine, nitrobenzylthioguanosine, and dipyridamole. Exposure of S49 membrane preparations to UV light in the presence of [3H]nitrobenzylthioinosine resulted in the covalent radiolabeling of a membrane protein(s) which migrated on sodium dodecyl sulfate-polyacrylamide gels with an apparent Mr of 45,000 to 66,000. Labeling of this protein was abolished in the presence of nitrobenzylthioguanosine and markedly reduced in the presence of adenosine and dipyridamole. AE1 membrane proteins were not covalently labeled under these conditions.

Topics: Adenosine; Affinity Labels; Animals; Binding, Competitive; Blood Proteins; Cell Line; Cell Membrane; Dipyridamole; Guanosine; Inosine; Lymphoma; Membrane Proteins; Mice; Nucleoside Transport Proteins; Thioinosine; Thionucleosides; Tritium

Cells of an adenosine-resistant clone (AE1) of S49 mouse lymphoma cells were compared with cells of the parental line with respect to (a) characteristics of nucleoside transport, (b) high affinity binding of the inhibitor of nucleoside transport, nitrobenzylthioinosine (NBMPR), and (c) the antiproliferative effects of the nucleoside antibiotics, tubercidin, arabinosyladenine and showdomycin. Rates of inward transport of uridine, thymidine, adenosine, 2'-deoxyadenosine, tubercidin, showdomycin, and arabinosyladenine in AE1 cells were less than 1% of those in cells of the parental S49 line. The inhibitor of nucleoside transport, NBMPR, reduced rates of inward nucleoside transport in S49 cells to levels comparable to those seen in the transport-defective mutant. S49 cells possessed high affinity sites that bound NBMPR (6.6 X 10(4) sites/cell, Kd = 0.2 nM), whereas site-specific binding of NBMPR to AE1 cells was not demonstrable, indicating that loss of nucleoside transport activity in AE1 cells was accompanied by loss of the high affinity NBMPR binding sites. Relative to S49 cells, AE1 cells were resistant to the antiproliferative effects of tubercidin and showdomycin, but differences between the two cell lines in sensitivity toward arabinosyladenine were minor, suggesting that nucleoside transport activity was required for cytotoxicity of tubercidin and showdomycin, but not for that of arabinosyladenine.

Topics: Adenosine; Animals; Anti-Bacterial Agents; Biological Transport; Cell Division; Cell Line; Clone Cells; Inosine; Kinetics; Lymphoma; Mice; Neoplasms, Experimental; Ribonucleosides; Thioinosine

A series of 2'-O-acyl derivatives of 6-thioinosine cyclic 3',5'-phosphate (6-HS-cRMP) were prepared and examined for their cytotoxic effects on S49 mouse lymphoma cells which were deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRTase). Cytotoxicity increased with the lipophilicity of the acyl group to a lowest EC50 of 65 micrometer for the 2'-O-palmityl derivative. Addition of a mutation in the gene for cAMP-dependent protein kinase to the HGPRTase-deficient cell line confers resistance to 2'-O-butyryl-cAMP but not to 2'-O-butyryl-6-HS-cRMP, indicating that the latter does not exert its toxic effect via activation of protein kinase. The time course of cell kill by 2'-O-palmityl-6-HS-cRMP resembled that of 6-mercaptopurine and not that of cyclic AMP in these cells. The data suggest that the intact cyclic nucleotides are penetrating the cells and being converted, by phosphodiesterase action and deacylation, to the first toxic metabolite of 6-mercaptopurine, thioinosinic acid.

Topics: Animals; Antineoplastic Agents; Cyclic AMP; Drug Resistance; Hypoxanthine Phosphoribosyltransferase; Inosine; Lymphoma; Mice; Neoplasms, Experimental; Nucleotides, Cyclic; Thioinosine; Thionucleotides; Time Factors