levoleucovorin and Leukemia--Erythroblastic--Acute

We previously identified two membrane-bound folate binding proteins, FBP1 and FBP2, in murine L1210 leukemia cells. We now report on the development of two variant murine erythroleukemia cell lines that were used for direct comparison and biochemical characterization of the two murine folate binding proteins. Based on the results of northern analysis and the mobilities of affinity-labeled proteins on polyacrylamide gels, these cell lines exhibit specific up-regulated expression of FBP1 or FBP2. The affinities of the folate binding proteins for various (anti)folates were determined based upon the ability of the compounds to inhibiting of [3H]folic acid. The two proteins exhibited considerably different affinities and stereospecificities and, in general, FBP2 consistently bound each test compound with lesser affinity than FBP1. Both proteins displayed greatest affinity for folic acid, 5-methyltetrahydrofolate, and the antifolates CB3717 and 5,10-dideazatetrahydrofolate (DDATHF). Conversely, the proteins exhibited poor affinity for the dihydrofolate reductase inhibitors methotrexate and aminopterin. For 5-formyltetrahydrofolate, FBP1 had high affinity for the (6S) diastereoisomer, whereas FBP2 showed preference for the non-physiologic (6R) diasterceoisomer. The binding properties of FBP1 and FBP2 overexpressed in these cell lines closely paralleled those of their respective human homologs. These lines provide a model system in which to examine the biochemical characteristics of the individual folate binding proteins without the potential problems associated with expression of proteins in dissimilar cell lines.

Topics: Amidohydrolases; Animals; Biological Transport; Carrier Proteins; Clone Cells; Folate Receptors, GPI-Anchored; Folic Acid; Gene Expression; Humans; Leucovorin; Leukemia, Erythroblastic, Acute; Mice; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Receptors, Cell Surface; Stereoisomerism; Tetrahydrofolates; Tumor Cells, Cultured

The direct effect of selected cytostatic drugs on natural killer (NK) cell activity was evaluated. Peripheral blood mononuclear cells from healthy donors were tested for their cytolytic activity in vitro in the presence of adriamycin, methotrexate, leucovorin, vincristin, cytosine arabinoside and teniposide. Most of the tested cytostatic drugs did not show to be active at concentrations comparable to their plasma level. However, diluents of some preparations (cytosine arabinoside, teniposide) containing organic solutions and stabilizing additives (e.g. benzyl alcohol) suppressed the NK activity more than chemotherapeutic agents alone. Thus teniposide, containing such additives, inhibited NK activity already at 5 mg/ml, while its peak plasma concentration was 23.8 mg/ml. The inhibitory concentrations of teniposide did not affect the target binding of effector cells and the expression of 14 tested leukocyte differentiation markers. This implies that a postbinding step of the lytic process was altered by the preparation. Likewise, no inhibition of lectin dependent cellular cytotoxicity by teniposide and its diluent was observed, suggesting that the lectin may substitute the missing lytic signal.

Topics: Antigens, Differentiation; Antineoplastic Agents; Benzyl Alcohol; Benzyl Alcohols; Cytarabine; Cytotoxicity Tests, Immunologic; Dose-Response Relationship, Drug; Doxorubicin; Fluorescent Antibody Technique; Humans; In Vitro Techniques; Killer Cells, Natural; Leucovorin; Leukemia, Erythroblastic, Acute; Methotrexate; Teniposide; Vincristine

A K562 human erythroleukemia line (designated K562.4CF) was selected for increased tetrahydrofolate cofactor transport in a growth-limiting concentration (0.4 nM) of (6R,S)-5-formyltetrahydrofolate. K562.4CF cells exhibited elevated methotrexate uptake relative to parental cells, attributable to a 10-fold increased influx Vmax. The rate of methotrexate efflux in K562.4CF cells was somewhat increased (55%) as well. The transport system in K562.4CF cells had similar and high apparent binding affinities for methotrexate and 5-formyltetrahydrofolate and a markedly reduced affinity for folic acid, properties typically associated with the "classical" methotrexate/tetrahydrofolate cofactor transporter in tumor cells. Methotrexate uptake in K562.4CF cells decreased substantially under nonselective conditions; high levels of transport were restored in 0.4 nM 5-formyltetrahydrofolate. Treatment of parental and K562.4CF cells with N-hydroxysuccinimide methotrexate inhibited methotrexate influx. N-Hydroxysuccinimide-[3H]methotrexate (700 nM) radiolabeled a broadly migrating band at Mr 76,000-85,000. Incorporation from N-hydroxysuccinimide-[3H]methotrexate into this band was increased 7-fold in K562.4CF over parental cells and was blocked by unlabeled methotrexate, (6S)-5-formyltetrahydrofolate, or, to a lesser extent, folic acid. Whereas incubation with endoglycosidase F had no effect on the electrophoretic migration of the labeled protein, treatment with endoglycosidase F and glycopeptidase F, or endo-beta-galactosidase, reduced the apparent molecular weight to Mr approximately 52,000 or approximately 58,000, respectively. These results suggest that the high-affinity transporter in K562.4CF cells is an N-linked glycoprotein containing internal beta-galactosidic linkages in, or immediately after, unbranched poly-N-acetyllactosamine sequences. Differences in the level of glycosylation may, in part, account for the disparity in the apparent sizes of the homologous folate transport proteins from human and murine cells.

Topics: Affinity Labels; Biological Transport; Carrier Proteins; Cell Membrane; Humans; Kinetics; Leucovorin; Leukemia, Erythroblastic, Acute; Membrane Glycoproteins; Methotrexate; Structure-Activity Relationship; Tumor Cells, Cultured

Methotrexate (MTX) is metabolized intracellularly to MTX-polyglutamates (MTX-PGs), which markedly inhibit several folate-dependent enzymes. Polyglutamation defect, therefore, is one of the important factors in drug resistance. In this study, reversal of MTX cytotoxicity by l-leucovorin (l-LV) was investigated using normal human bone marrow granulocyte progenitor cells (G-CFCs), and MTX-sensitive and -resistant leukemic K562 cell lines; the latter showed diminished polyglutamation. Cytotoxicity of 10(-7)M MTX to G-CFCs was completely reversed by an equimolar concentration of l-LV, but with higher MTX concentrations, relatively more l-LV was required. The reversal of MTX cytotoxicity by l-LV was more effective against bone marrow cells than MTX-sensitive K562 cells; this reversal seemed to be correlated to the total intracellular MTX levels as well as MTX-PG formation (low in bone marrow cells and high in K562 cells). When MTX-sensitive and -resistant K562 cells were incubated with MTX under conditions in which the total intracellular MTX levels of both cells were similar, successful reversal of MTX toxicity by l-LV was demonstrated in MTX-resistant cells, but not in MTX-sensitive cells, suggesting that an increase of MTX-PG formation in MTX-sensitive cells may explain the failure of l-LV to overcome MTX cytotoxicity. In addition to competitive reversal of MTX cytotoxicity by LV, noncompetitive reversal relating to variable formation of MTX-PGs is suggested to be another important factor in the mechanism of the reversal of MTX cytotoxicity by LV.

Topics: Antineoplastic Combined Chemotherapy Protocols; Bone Marrow; Dose-Response Relationship, Drug; Drug Resistance; Granulocytes; Humans; In Vitro Techniques; Leucovorin; Leukemia, Erythroblastic, Acute; Methotrexate; Polyglutamic Acid

The effects of 5-fluorouracil (5-FUra), in combination with various differentiation inducers on the growth and differentiation of mouse erythroleukemia (MEL) cells were investigated. The cells were first treated with 5-FUra, washed, and then treated with various concentrations of differentiation inducers: hexamethylene bisacetamide (HMBA), dimethyl sulfoxide (DMSO), and N-methylformamide. Pretreatment with 5-FUra, shown here to be a weak inducer of MEL cell differentiation, enhanced the subsequent HMBA induction of differentiation. The three inducers of differentiation markedly inhibited cell growth and increased cell death in a dose- and time-dependent manner if given immediately after cells were exposed to 5-FUra. In contrast, 5-FUra at similar concentrations inhibited cell growth, but only slightly increased cell death, while inducers without 5-FUra had little effect on cell growth or viability. When placed in fresh drug-free medium for 6 days following drug treatments, the cells completely recovered from the growth inhibition of 5-FUra as a single agent, whereas in cells previously treated with only HMBA there was a inhibition of cell growth without loss of viability. In contrast, a profound and prolonged growth inhibition with 98% cell death occurred in cells previously treated with 5-FUra followed by HMBA. The enhancement of 5-FUra cytotoxicity appeared to be directly related to the degree of differentiation and to biochemical events that occur during the commitment to terminal cell division induced by N-methylformamide, DMSO, or HMBA. An increase in Okazaki fragments was found in MEL cells treated with HMBA or DMSO when committed to terminal cell division. DNA breaks also follow 5-FUra treatment (A. Yoshioka et al., J. Biol. Chem., 262: 8235-8241, 1987) and may be the events that lead to cell death. The marked increase in cell death resulting from 5-FUra/HMBA treatment may be, at least partly, a consequence of increased DNA breaks due to 5-FUra followed by inhibition of DNA repair which is known to occur following the HMBA or DMSO induction of differentiation and commitment to terminal cell division. This combined sequential cytotoxic-differentiation therapy resulting in synergistic cytotoxicity and differentiation may be the basis of a new approach to cancer therapy and may aid in reducing the amounts of chemotherapeutic agents required for effective treatment, while maintaining or even increasing their therapeutic effects.

Topics: Acetamides; Animals; Cell Count; Cell Differentiation; Cell Division; Dexamethasone; Dimethyl Sulfoxide; DNA Repair; DNA, Neoplasm; Drug Synergism; Drug Therapy, Combination; Fluorouracil; Formamides; Leucovorin; Leukemia, Erythroblastic, Acute; Mice; Time Factors; Tumor Cells, Cultured

Topics: Animals; Antimetabolites; Cell Count; Cell Division; Cell Line; Deoxyuridine; DNA, Neoplasm; Dose-Response Relationship, Drug; Drug Interactions; Fluorouracil; Folic Acid; Leucovorin; Leukemia, Erythroblastic, Acute; Mice; Vitamin E; Vitamin K

Cultures of Friend erythroleukemia cells were subjected to the antibiotics trimethoprim (T) and sulfamethoxazole (S) at levels equal to or below the usual therapeutic range. At T 8 microgram/ml and S 40 microgram/ml, cell growth was arrested, cells appeared megaloblastic, and the examination of cell-cycle distribution by flow microfluorimetry revealed arrest in S phase. With a tenfold reduction in drug levels (T, 08 microgram/ml; S, 4 microgram/ml) cell growth was less markedly inhibited, morphology remained megaloblastic, and S-phase block was still dramatic. A further tenfold reduction (T, 0.08 microgram/ml; S, 0.4 microgram/ml), well below effective antibacterial levels, allowed normal cell growth and morphology but DNA synthesis was still inhibited. Additions of folinic acid at 100 ng/ml averted all drug effects. Thus T/S can affect cell replication even at levels well below those usually employed and could prolong the rate of recovery of hematopoietic cells in the myelosuppressed patient.

Topics: Cell Transformation, Neoplastic; Cells, Cultured; DNA; Dose-Response Relationship, Drug; Erythrocyte Volume; Friend murine leukemia virus; Leucovorin; Leukemia, Erythroblastic, Acute; Megaloblasts; Sulfamethoxazole; Trimethoprim

Topics: Folic Acid; Humans; Leucovorin; Leukemia, Erythroblastic, Acute; Leukemia, Myeloid, Acute