methylcellulose and Precursor-Cell-Lymphoblastic-Leukemia-Lymphoma

The γ-glutamyl hydrolase (GGH) gene plays an important role in methotrexate (MTX) metabolism, ensuring that MTX polyglutamates (MTX-(Glu)(n)) could be converted back into MTX. Accumulation of MTX-(Glu)(n) is a problem in MTX therapy. SNP 452 C>T has been reported to associate with lower catalytic activity and higher accumulation of long-chain MTX-(Glu)(n) in patients treated with higher doses of MTX treatment. We propose and establish a simple and effective CE method for detecting SNP in GGH gene. The DNA samples after amplification were analyzed by SSCP-CE method. The CE conditions were generated by using 1× TBE buffer containing 1.5% w/v hydroxypropyl methyl cellulose under reverse polarity at 25°C. This method was applied to detect genotyping of acute lymphoblastic leukemia patients receiving MTX treatment. The results were confirmed by DNA sequencing with good agreement. Concentrations of MTX-(Glu)(n) in whole blood were analyzed by on-line stacking CE method. MTX-(Glu)(n) levels and genotypes in GGH gene of acute lymphoblastic leukemia patients were evaluated. The SSCP-CE method was found to be feasible for SNP screening in the GGH gene.

Topics: Antimetabolites, Antineoplastic; Electrophoresis, Capillary; gamma-Glutamyl Hydrolase; Genotype; Humans; Hypromellose Derivatives; Methotrexate; Methylcellulose; Polymorphism, Single Nucleotide; Polymorphism, Single-Stranded Conformational; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Temperature

In a systematic effort to identify and develop effective anticancer agents, four oxovanadium(IV) complexes with 1,10-phenanthroline (Phen) or 4,7-dimethyl-1,10-phenanthroline (Me2-Phen) as ligand(s) were synthesized and characterized. Among the four oxovanadium(IV) complexes synthesized, the crystal structure of the bis(phenanthroline)oxovanadium(IV) complex bis(1,10-phenanthroline)sulfatooxovanadium(IV) ([VO(SO4)(Phen)2], compound 1) has been determined. Compound 1 crystallized in the space group P2(1)/n with unit cell parameters a = 14.2125(17) A, b = 10.8628(13) A, c = 20.143(2) A, alpha = 90 degrees, beta = 102.569(2) degrees, gamma = 90 degrees, V = 3035.3(6) A3, and Z = 4. The refinement of compound 1 by full-matrix least-squares techniques gave an R factor of 0.0785 for 4356 independent reflections. The structure contains two enantiomorphous molecules, lambda and delta, which are related by an inversion center. Compound 1 exhibited 3.5-fold more potent cytotoxic activity against NALM-6 human leukemia cells than the mono(phenanthroline)oxovanadium(IV) complex (diaqua)(1,10-phenanthroline)sulfatooxovanadium(IV) ([VO(SO4)(Phen)(H2O)2], compound 2) (IC50 values: 0.97+/-0.10 microM versus 3.40+/-0.20 microM: P=0.0004). Methyl substitution in the phenanthroline ligand enhanced the anti-leukemic activity of the mono(phenanthroline)oxovanadium(IV) complex 4.4-fold (IC50 values: 0.78+/-0.10 microM, compound 4, versus 3.40+/-0.20 microM, compound 2; P=0.0003) and the anti-leukemic activity of the bis(phenanthroline)oxovanadium(IV) complex 5.7-fold (IC50 values: 0.17+/-0.02 microM, compound 3, versus 0.97+/-0.10 microM, compound 1; P=0.001). The leading oxovanadium compound, bis(4,7-dimethyl-1,10-phenanthroline)sulfatooxovanadium(IV) ([VO(SO4)(Me2-Phen)2], compound 3) triggered the production of reactive oxygen species (ROS) in human leukemia cells, caused G1-arrest and inhibited clonogenic growth at nanomolar concentrations.

Topics: Antineoplastic Agents; Cell Cycle; Cell Division; Cell Survival; Crystallography, X-Ray; Dose-Response Relationship, Drug; Humans; Inhibitory Concentration 50; Ligands; Methylcellulose; Models, Chemical; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Reactive Oxygen Species; Time Factors; Tumor Cells, Cultured; Vanadates

We studied bone marrow separated with 0.05% methylcellulose and cryopreservatized with liquid nitrogen. The separation time was 43 minutes. The collection rates of nuclear cells and CFU-GM were 79.0 +/- 5.10% and 93.0 +/- 3.10% in normal marrow samples and 83.4 +/- 15.45% and 91.0 +/- 8.32% in those of acute leukemia patients. The rate of residual erythocytes was 12.5% and the bone marrow volume could be reduced by 55.2%. Application of this technique to autologous bone marrow transplantation in four patients with acute leukemia reconstituted all of their hemopoietic functions and no toxic side effect was found after transplantation.

Topics: Bone Marrow; Bone Marrow Transplantation; Cell Separation; Cryopreservation; Humans; Leukemia, Myeloid, Acute; Methylcellulose; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Transplantation, Autologous

In studies of in vitro leukemic clonogenic cells it is of importance to determine the cell lineage of individual clusters grown in culture. A method is described for the in-situ identification of leukemic cell clusters in methylcellulose cultures. Whole cultures are dried and incubated with various monoclonal antibodies, followed by incubation with beads coated with secondary antibody. Clusters containing antibody-positive cells are heavily labeled with beads which simplifies the recognition and scoring of clusters using normal light microscopy. This method has general applications and can also be used to identify normal myeloid and lymphoid clusters depending on the availability of lineage specific monoclonal antibodies.

Topics: Bone Marrow; Bone Marrow Cells; Child; Humans; Immunophenotyping; Methylcellulose; Neoplastic Stem Cells; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Tumor Cells, Cultured; Tumor Stem Cell Assay