thioinosine and Cell-Transformation--Neoplastic

The experience with myeloproliferative diseases (MPD) in children of a large pediatric hematology-oncology service during a 20-year period is reviewed. Twenty-seven patients with myeloproliferative diseases were treated, six with the juvenile type of chronic myelogenous leukemia (CML), 10 with the adult type of CML, three with familial MPD, one with unclassifiable MPD, and seven with acute leukemia in whom myelofibrosis was either a prodromal or terminal event. The literature is reviewed with particular emphasis regarding the relationships between the juvenile type of CML and monocytic leukemia, the adult type of CML and acute nonlymphocytic leukemia, and the relationship of myelofibrosis and myeloid metaplasia to the acute leukemias. New therapeutic approaches are needed in this heterogenous but interrelated group of disorders.

Topics: Adolescent; Adrenal Cortex Hormones; Busulfan; Cell Transformation, Neoplastic; Child; Child, Preschool; Chromosomes, Human, 21-22 and Y; Female; Fetal Hemoglobin; Humans; Infant; Leukemia, Erythroblastic, Acute; Leukemia, Myeloid; Male; Methotrexate; Myeloproliferative Disorders; Primary Myelofibrosis; Thioinosine

(18)F-3'-Deoxy-3'-fluorothymidine ((18)F-FLT) is a PET tracer that accumulates in proliferating tissues. The current study was undertaken to determine whether equilibrative nucleoside transporter 1 (ENT1) is important for (18)F-FLT uptake in normal tissues and tumors.. ENT1-knockout (ENT1(-/-)) mice were generated and compared with wild-type (ENT1(+/+)) mice using small-animal (18)F-FLT PET. In addition, ENT1(+/+) mice were also injected with the ENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside phosphate (NBMPR-P) at 1 h before radiotracer injection, followed by (18)F-FLT small-animal PET. Tissues of interest were analyzed for thymidine kinase 1 and nucleoside transporters by immunoblotting and immunohistochemistry, respectively, and plasma thymidine levels were analyzed by liquid chromatography-mass spectrometry. Human lung carcinoma A549 cells were stably transfected with pSUPER-producing short-hairpin RNA against human ENT1 (hENT1) or a scrambled sequence with no homology to mammalian genes (A549-pSUPER-hENT1 and A549-pSUPER-SC, respectively). Cultured transfected cells were characterized for hENT1 transcript levels and (18)F-FLT uptake using real-time polymerase chain reaction and (3)H-FLT uptake assays, respectively. Transfected A549 cells were grown as xenograft tumors in NIH-III mice, which were analyzed by (18)F-FLT small-animal PET.. Compared with noninjected ENT1(+/+) mice, ENT1(+/+) mice injected with NBMPR-P and ENT1(-/-) mice displayed a reduced percentage injected dose per gram (%ID/g) for (18)F-FLT in the blood (84 and 81%, respectively) and an increased %ID/g for (18)F-FLT in the spleen (188 and 469%, respectively) and bone marrow (266 and 453%, respectively). ENT1(-/-) mice displayed 1.65-fold greater plasma thymidine levels than did ENT1(+/+) mice. Spleen tissue from ENT1(+/+) and ENT1(-/-) mice displayed similar thymidine kinase 1 protein levels and significant concentrative nucleoside transporter 1 and 3 staining. Compared with A549-pSUPER-SC cells, A549-pSUPER-hENT1 cells displayed 0.45-fold hENT1 transcript levels and 0.68-fold (3)H-FLT uptake. Compared with A549-pSUPER-SC xenograft tumors, A549-pSUPER-hENT1 xenograft tumors displayed 0.76-fold %ID/g values (ex vivo gamma-counts) and 0.65-fold maximum standardized uptake values (PET image analysis) for (18)F-FLT uptake at 1 h after tracer injection.. Loss of ENT1 activity significantly affected (18)F-FLT biodistribution in mice and (18)F-FLT uptake in xenograft tumors, suggesting that nucleoside transporters are important mediators of (18)F-FLT uptake in normal and transformed cells.

Topics: Animals; Biological Transport; Cell Line, Tumor; Cell Transformation, Neoplastic; Dideoxynucleosides; Equilibrative Nucleoside Transporter 1; Female; Gene Knockdown Techniques; Gene Knockout Techniques; Humans; Mice; Mice, Knockout; Neoplasms; Positron-Emission Tomography; RNA, Messenger; RNA, Small Interfering; Spleen; Thioinosine; Thymidine; Thymidine Kinase; Transfection

Adenosine, an important neuromodulatory compound in the brain and retina, is a potent vasodilator in most vascular beds throughout the body. Its actions are potentiated by inhibitors of nucleoside transport into cells. Knowledge of the existence of specific adenosine uptake systems in mammalian retina and the inhibition of the uptake by nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport, raises the possibility that the associated nucleoside transport system may be of pharmacological importance in retinal function. We have characterized the binding of the nucleoside transporter probe, [3H]NBMPR, to a cultured human retinal cell line established by transfection of SV-40 T antigen plasmid-DNA. The binding was specific, saturable and reversible. Scatchard analysis of the saturation data revealed that NBMPR binds to a homogeneous population of high affinity binding sites (KD = 0.65 +/- 0.22 nM; Bmax = 466 +/- 157 fmol/mg protein) characteristically similar to the binding sites in human retinal tissue (KD = 0.32 +/- 0.01 nM; Bmax = 292 +/- 41 fmol/mg protein). Selected compounds inhibited the binding in the cell line and retinal tissue with the same rank order of potency, suggesting that the transporters in the cell line and retinal tissue are similar. The data showed that the cell line is a useful model for the study of nucleoside transporter function in human retina.

Topics: Adenosine; Affinity Labels; Antigens, Polyomavirus Transforming; Blood Proteins; Carrier Proteins; Cell Transformation, Neoplastic; Fetus; Humans; Membrane Proteins; Nerve Tissue Proteins; Nucleoside Transport Proteins; Retina; Thioinosine; Transfection; Tumor Cells, Cultured

Important cellular nutrients, including nucleosides and hexose sugars, are rapidly taken up by cells, largely through mediated carrier systems. The present study examined nucleoside and hexose transport activity in normal Rat-2 fibroblasts and clonal derivatives that expressed either the wild-type (C10) or a temperature-sensitive mutant (NA9) form of v-fps, a transforming protein-tyrosine kinase. Initial uptake rates (transport) of adenosine, thymidine, 3-O-methylglucose and 2-deoxyglucose were greater in v-fps-transformed cells than in normal cells. Elevated transport rates were seen in cells that expressed the temperature-sensitive mutant v-fps only after growth at a temperature that was permissive for protein-tyrosine kinase activity. Nucleoside transport rates declined with increasing cell density in both normal and v-fps transformed cells. Analysis of the sensitivity of adenosine transport to inhibition by nitrobenzylthioinosine (NBMPR) indicated that Rat-2 fibroblasts, like many other rat cell types, possess at least two nucleoside transport systems, which can be distinguished by differences in sensitivity to NBMPR. Although both transport activities were elevated in v-fps-transformed cells, a greater increase was seen in the NBMPR-sensitive component than in the NBMPR-insensitive component. Mass law analysis of the binding of [3H]NBMPR indicated that transformed cells had either the same number (NA9) or a smaller number (C10) of NBMPR-binding sites than normal cells, and photolabelling of membrane proteins with [3H]NBMPR identified polypeptides with similar electrophoretic mobilities (55-75 kDa) in both normal and transformed cells. Thus transformation by v-fps resulted in an increase in NBMPR-sensitive transport activity which was not related to either the number of NBMPR-binding sites or the apparent molecular mass of NBMPR-binding polypeptides.

Topics: 3-O-Methylglucose; Adenosine; Affinity Labels; Animals; Biological Transport; Cell Line; Cell Line, Transformed; Cell Membrane; Cell Transformation, Neoplastic; Deoxyglucose; Fibroblasts; Fusion Proteins, gag-onc; Genes, gag; Kinetics; Methylglucosides; Nucleosides; Protein-Tyrosine Kinases; Rats; Thioinosine; Thymidine

Although cytosine arabinoside (araC) can induce a remission in a majority of patients presenting with acute myeloblastic leukemia (AML), a minority fail to respond and moreover the drug has less effect in acute lymphoblastic leukemia (ALL). The carrier-mediated influx of araC into purified blasts from patients with AML, ALL, and acute undifferentiated leukemia (AUL) has been compared to that of normal lymphocytes and polymorphs. Blasts showed a larger mediated influx of araC than mature cells, since mean influxes for myeloblasts and lymphoblasts were 6- and 2.3-fold greater than polymorphs and lymphocytes, respectively. Also, the mean influx for myeloblasts was fourfold greater than the mean for lymphoblasts. The number of nucleoside transport sites was estimated for each cell type by measuring the equilibrium binding of [(3)H]nitrobenzylthioinosine (NBMPR), which inhibits nucleoside fluxes by binding with high affinity to specific sites on the transport mechanism. The mean binding site numbers for myeloblasts and lymphoblasts were 5- and 2.8-fold greater, respectively, than for the mature cells of the same maturation series. The mean number of NBMPR binding sites for myeloblasts was fourfold greater than for lymphoblasts. Patients with AUL were heterogeneous since blasts from some gave values within the myeloblastic range and others within the lymphoblastic range. The araC influx correlated closely with the number of NBMPR binding sites measured in the same cells on the same day. Transport parameters were measured on blasts from 15 patients with AML or AUL who were then treated with standard induction therapy containing araC. Eight patients entered complete remission, while seven failed therapy, among whom were the three patients with the lowest araC influx (<0.4 pmol/10(7) cells per min) and NBMPR binding (<3,000 sites/cell) for the treated group. In summary, myeloblasts have both higher araC transport rates and more nucleoside transport sites than lymphoblasts and this factor may contribute to the greater sensitivity of AML to this drug. AraC transport varied >10-fold between leukemic blasts and normal leukocytes, but transport capacity related directly to the number of nucleoside transport sites on the cell. Finally, low araC transport rates or few NBMPR binding sites on blasts were observed in a subset of patients with acute leukemia who failed to achieve remission with drug combinations containing araC.

Topics: Acute Disease; Adolescent; Adult; Aged; Binding Sites; Bone Marrow Cells; Carrier Proteins; Cell Transformation, Neoplastic; Child; Child, Preschool; Cytarabine; Humans; Infant; Leukemia; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Leukocytes; Middle Aged; Nucleosides; Thioinosine; Time Factors

Topics: Adult; Aged; Cell Transformation, Neoplastic; Female; Humans; Leukemia, Myeloid; Male; Middle Aged; Prednisolone; Thioinosine; Vincristine

Topics: Acute Disease; Cell Transformation, Neoplastic; Cytarabine; Daunorubicin; Humans; Leukemia; Prednisolone; Thioinosine

The kinetics of uridine uptake in growing, quiescent and serum-activated hamster embryo cells are investigated. The maximum velocity of uridine uptake in growing hamster embryo cells, is lower than in the methylcholanthrene transformed hamster cell line (MCT). This kinetic constant is further reduced in quiescent cells. The Km values in growing and in quiescent hamster embryo cells, as well as in MCT cells are of the same magnitude. Distinct alterations in the pattern of inhibition by nitrobenzyl 6-mercaptoinosin (NBMI) are detected as growing hamster embryo cells become quiescent. In quiescent cells the maximum level of inhibition is lower and the apparent Ki value for the inhibition is much higher. These changes are due to the lower apparent K'm values of NBMI-bound carriers and to the slower rate of formation of the carrier-inhibitor complex. The changes in the kinetic properties of the carriers are partly reversed by serum-activation. The number of inhibitor binding sites (i.e. nucleoside carriers) does not increase by serum-stimulation of quiescent cells (0.36 and 0.34-10(5) sites/cell in quiescent and serum-stimulated cells, respectively). It is implied that the reduction in uridine transport in quiescent cells is probably due to changes in the turnover of the carriers. These changes may be connected with the observed alterations in the properties of carriers or their immediate environment in quiescent cells.

Topics: Animals; Biological Transport, Active; Blood; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cricetinae; Kinetics; Methylcholanthrene; Phenylmercury Compounds; Thioinosine; Uridine