methylcellulose and Leukemia--Myeloid

Haematopoietic stem cells are capable of developing colonies of differentiated granulocytes in a semi-stable agar environment under corresponding experimental conditions. Number and size of aggregations developing from a single stem cell in each case called "in culture Colony Forming Unit (CFU-c)" enable conclusions to be made about the functional ability of the cultivated human or animal bone marrow. The method is applied in investigating the kinetics in the haematopoietic system and, in addition, it is used for diagnostics, therapy and control of the course in haematological diseases as well as to check the proliferous ability of cryopreserved bone marrow cells. As to the standardization of the procedure the instability of the active principle of the foetal calf serum as well as the essential colony stimulating factor represent limiting items at present.

Topics: Agar; Animals; B-Lymphocytes; Bone Marrow Cells; Cell Differentiation; Cell Division; Cells, Cultured; Chick Embryo; Colony-Stimulating Factors; Culture Techniques; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Methylcellulose; Mice; T-Lymphocytes; Thymus Gland

Regulatory molecules that affect the growth culture of blast cells from acute myeloblastic leukemia (AML) may also alter drug sensitivity, a phenomenon that may be called regulated drug sensitivity. Previous studies have shown: (i) blast cells exposed to retinoic acid before cytosine arabinoside (Ara-C) usually show increased sensitivity, but after some retinoic acid exposure times, sensitivity may be decreased; (ii) factor-sensitive or responsive blasts cultured with granulocyte colony-stimulating factor (G-CSF) are regularly more Ara-C-sensitive than when cultured with granulocyte-macrophage CSF (GM-CSF). This paper is concerned with the effects of schedule on drug sensitivity as regulated by either retinoic acid or the myelopoietic growth factors, G-CSF and GM-CSF. We measured the effects of retinoic acid on the sensitivity of blasts cells from the two continuous AML lines to Ara-C or arabinofuranosyl 5-azacytosine (Ara-AC). Cells from seven patients with AML were tested for Ara-C sensitivity in conjunction with retinoic acid. The cells were treated with retinoic acid before or after administration of the drug. Both increases and decreases in Ara-C sensitivity were seen for both schedules. Consistent increases in Ara-C sensitivity were obtained when retinoic acid was included in the methylcellulose cultures used to determine clonogenic cell recovery at each drug dose. In studies of growth factors, a single factor-dependent cell line (OCI/AML-5) was used to compare the effects of G-CSF and GM-CSF on Ara-C sensitivity. An experimental design was used that permitted factors to present in culture for 24 h before Ara-C, during the next 24 h period with the drug, for a subsequent day in suspension without drug, and during the 5-7 days required for colony formation in methylcellulose cultures. G-CSF and GM-CSF were most effective in increasing or decreasing Ara-C, respectively, when the factor under test was included in the methylcellulose cultures. Thus, like retinoic acid, growth factors influenced drug sensitivity when they were present after the drug had been removed. These data, therefore, are compatible with the hypothesis that repair mechanism may contribute to regulated drug sensitivity.

Topics: Acute Disease; Aged; Cell Survival; Cytarabine; Female; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; In Vitro Techniques; Leukemia, Myeloid; Male; Methylcellulose; Middle Aged; Tretinoin; Tumor Cells, Cultured

Deferoxamine (DFO) possesses antiproliferative activity against mitogen-stimulated lymphocytes, several tumor cell lines, and human leukemia and neuroblastoma cells. We have investigated its effects on the human myeloid leukemia lines HL-60, HEL, and U-937. In suspension culture, DFO causes a dose-dependent inhibition of proliferation of each cell line, with maximal inhibition observed at concentrations greater than 20 microM. These effects were prevented by cotreatment with iron salts and were at least partially reversible by removal of DFO from the culture system or addition of iron before 48 h of DFO exposure. Similar results were obtained in methylcellulose cultures of leukemic cells, with complete abolition of cell aggregates at day 7 in concentrations of 20 microM DFO or higher. DFO treatment caused a dose- and time-related decrease in DNA synthesis as measured by [3H]thymidine uptake, which was also reversed by treatment with iron salts. DFO caused slight reduction in RNA synthesis and did not affect protein synthesis. DFO caused significant antiproliferative effects on three myeloid leukemia cell lines, associated with inhibition of DNA synthesis, with in vitro effects observed at concentrations attainable in vivo. Evaluation of the antileukemic properties of DFO should continue.

Topics: Antineoplastic Agents; Cell Division; Deferoxamine; DNA, Neoplasm; Drug Screening Assays, Antitumor; Humans; Leukemia, Myeloid; Methylcellulose; Neoplasm Proteins; RNA, Neoplasm; Time Factors; Tumor Cells, Cultured

Basophilia has been reported to indicate an accelerated phase of chronic myeloid leukemia (CML), heralding a poor prognosis. We have studied 47 patients with chronic-phase CML by basophil growth and differentiation assays in vitro, demonstrating an association between basophil growth index (BGI) and clinical time to blast crisis as well as overall survival. In addition to confirming an association between positive BGI and phase of CML in a larger group of patients, a positive BGI predicted death or blast crisis within 2 years' study of chronic phase CML (p less than 0.01), with a sensitivity of 78%, specificity, 81%; the positive predictive value of a positive test, 64%; and a negative predictive value of a negative test, 89%. The survival experience of the 22 evaluable patients with chronic-phase CML and a positive BGI was significantly worse than the survival of the 19 patients with a negative BGI (p less than 0.0001). At 1, 2, and 3 years the proportion of surviving patients with a positive BGI was 0.64, 0.32, and 0.23, respectively, compared with 1.00, 0.90, and 0.79 for those with a negative BGI. The median survival of chronic phase CML patients with a positive test at diagnosis (n = 14) was 27 months versus 54 months for those with a negative diagnosis (n = 14) (p less than 0.05). These findings emphasize the prognostic utility of basophil growth assays in CML and suggest a molecular relationship between leukemic transformation and basophil lineage expression.

Topics: Basophils; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Colony-Forming Units Assay; Female; Hematopoietic Stem Cells; Histamine; Humans; Leukemia, Myeloid; Male; Methylcellulose; Prognosis

Monoclonal antibodies (MoAbs) to myeloid differentiation antigens have a potential use in purging bone marrow of leukemia cells in autologous bone marrow transplantation (ABMT) therapy for acute myelogenous leukemia (AML). Because the efficiency of purging by MoAb and complement (C) is important to the success of ABMT, we have designed an assay to determine optimal conditions for leukemia cell lysis. In order to mimic the conditions of remission bone marrow, normal buffy coat cells were mixed with cells from the HL-60 promyelocytic leukemia cell line at a concentration that approximated the normal-leukemia cell ratio found in remission marrow. The cell mixture was treated at variable times and temperatures in the presence of C and PM-81, an IgM MoAb that reacts with both normal granulocytes and monocytes as well as with HL-60 cells. PM-81 binds to the majority of cells from 90% of patients with AML yet does not react significantly with normal stem cell populations. Because of the potential use of PM-81 in ABMT, it seemed especially important to show that the antibody was capable of mediating cytotoxicity of HL-60 cells in the presence of an excess of antigen-positive cells. A clonogenic assay that permitted the growth of HL-60 cell colonies but not normal progenitor cells in methylcellulose cultures was used to measure the efficiency of HL-60 cell lysis. We found that under certain conditions, PM-81 was capable of removing the small percentage of HL-60 clonogenic cells admixed with normal buffy coat cells. This information was useful for determining the optimal conditions for purging bone marrow of leukemia cells for ABMT.

Topics: Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Antigens, Neoplasm; Cell Line; Cell Survival; Complement System Proteins; Culture Media; Dose-Response Relationship, Immunologic; Humans; Leukemia, Myeloid; Methylcellulose; Neoplastic Stem Cells; Stem Cells; Tumor Stem Cell Assay

Polyriboinosinic-polycytidylic acid with poly-L-lysine stabilized with carboxymethylcellulose [poly(I,C)-LC] augmented, in a dose- and time-dependent manner, secretion of colony-stimulating factor (CSF) by peritoneal macrophages (M phi) and bone marrow cells (BMC). Optimal effects were found after 2 days of in vitro culture of the cells with 50 micrograms/ml of poly(I,C)-LC or 14 h to 3 days after a single intraperitoneal injection of 1-2 mg/kg of poly(I,C)-LC into normal mice. The increase in CSF secretion by M phi and BMC was paralleled in vivo by an increase in serum CSF levels, followed by a rise in committed granulocyte and M phi progenitor cells (GM-CFU-C), nucleated BMC, and blood leukocytes of myelomonocytic origin. Poly(I,C)-LC at doses greater than 4 mg/kg, however, were strongly myelosuppressive. In vitro treatment of undifferentiated myelomonocytic leukemia cells from the WEHI-3B cell line with 10-1,000 micrograms/ml of poly(I,C)-LC resulted in a significant increase in CSF secretion by the leukemic cells and a concomitant inhibition of their proliferation. Incubation of cells from the WEHI-3B D+ subline, which differentiate in response to GM-CSF or G-CSF, with 50-100 micrograms/ml poly(I,C)-LC in agar cultures induced in approximately 45% of the leukemic colonies a differentiation into granulocytes and/or M phi. Poly(I,C)-LC, however, had no effect on differentiation of cells from the CSF unresponsive WEHI-3B D- subline. The CSF-inducing biological response modifier poly(I,C)-LC thus has the potential to stimulate growth and differentiation of normal, as well as differentiation of malignant myelopoietic progenitor cells.

Topics: Animals; Carboxymethylcellulose Sodium; Cell Line; Colony-Stimulating Factors; Hematopoiesis; Hematopoietic Stem Cells; Leukemia, Myeloid; Macrophages; Male; Methylcellulose; Mice; Mice, Inbred BALB C; Neoplastic Stem Cells; Poly I-C; Polylysine

Topics: Agar; Cell Aggregation; Cell Line; Colony-Forming Units Assay; Colony-Stimulating Factors; Culture Media; Humans; Leukemia, Myeloid; Methylcellulose; Time Factors

A patient with Ph1-positive chronic myelogenous leukemia (CML) who entered an erythroblastic transformation prior to the development of a typical myeloblastic crisis is described. In vitro methylcellulose cultures obtained at the time of erythroblastic transformation revealed that the erythroid progenitors were responsive to erythropoietin. Thus, similar to the findings in polycythemia vera, the erythroid progenitors in this case of erythroblastic transformation of CML retained responsiveness to erythropoietin in vitro.

Topics: Bone Marrow; Bone Marrow Cells; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; Erythroblasts; Erythrocytes; Erythropoietin; Female; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Methylcellulose; Middle Aged

Topics: Cells, Cultured; Culture Media; Humans; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Methylcellulose

Topics: Aneuploidy; Cell Division; Cells, Cultured; Chromosome Aberrations; Culture Media; Humans; Karyotyping; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Lymphocytes; Methylcellulose

Topics: Bone Marrow; Bone Marrow Cells; Cell Line; Culture Media; Culture Techniques; Embryo, Mammalian; Humans; Kidney; Leukemia, Lymphoid; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Leukocytes; Methylcellulose; Monocytes