Tumor promoters enhance myeloid and erythroid colony formation by normal mouse hemopoietic cells.

The diterpene tumor promoters enhance the proliferation in culture of myeloid and erythroid precursor cells from normal mouse hemopoietic tissues. The effect is observed only with those diterpenes that are tumor promoters, including 12-O-tetradecanoylphorbol 13-acetate (TPA); diterpenes that are inactive as tumor promoters are ineffective as stimulators of colony formation. Tumor promoters act synergistically with suboptimal concentrations of conditioned medium used as a source of colony-stimulating factor (CSF) to increase both the number and size of myeloid colonies. Formation of myeloid colonies is stimulated by tumor promoters even without addition of CSF. Both pure and mixed granulocyte/macrophage colonies develop; high concentrations (> 100 micrograms/ml) of TPA are more favorable for macrophage colony formation. In erythropoietin-stimulated cultures, tumor promoters enhance the development of relatively early and intermediate of erythroid precursors, whereas later erythroid precursors are unaffected.     

Probenecid inhibition of methotrexate cytotoxicity in mouse L1210 leukemia cells

The effect of probenecid on methotrexate cytotoxicity has been measured using mouse L1210 leukemia cells in vitro. Cytotoxic effects were measured using a soft agar cloning technique. Cell exposure to varying concentrations of methotrexate at fixed concentrations of probenecid resulted in increased cell survival when compared with exposure to methotrexate alone. Cell exposure to a fixed concentration of methotrexate and varying concentrations of probenecid showed this effect to be dependent on the concentration of probenecid. Intracellular methotrexate concentrations were unchanged by the presence of probenecid. However, there was an effect of probenecid on progression of cells through the cell cycle which would tend to decrease the number of cells susceptible to the cytotoxic effects of methotrexate.     

Inhibition of human erythroid colony formation by ceramide.

In previous studies, we have demonstrated that the inhibitory effects of tumor necrosis factor (TNF) and interleukin (IL)-1 on human erythroid colony formation are indirect and mediated by beta and gamma interferon (IFN), respectively, which act directly upon erythroid colony forming units (CFU-E). The in vitro inhibitory effect of gammaIFN but not betaIFN is reversed by exposure to high concentrations of recombinant human (rh) erythropoietin (EPO). Ceramide, a product of sphingomyelin hydrolysis, is a known mediator of apoptotic effects of TNF, IL-1, and gammaIFN. In this report, the effects of ceramide on CFU-E colony formation and its implication in the model described above are evaluated. Endogenous ceramide produced by exposure to bacterial sphingomyelinase (0.2-2.0 U/mL) and exogenous cell-permeable ceramide (C2-ceramide; 5 and 10 mM) significantly inhibited bone marrow CFU-E colony formation. This effect was reversed by the ceramide antagonist sphingosine-1-phosphate (S-1-P). Inhibition of CFU-E by rhgammaIFN, but not rhbetaIFN, was significantly reversed by S-1-P. rhEPO 10 U/mL reversed CFU-E inhibition by C2-ceramide 10 mM. Exposure of marrow cells to rhgammaIFN led to a 57% increase in ceramide content. The present study demonstrates that colony formation by human CFU-E is inhibited by endogenous and exogenous ceramide, and that inhibition by rhgammaIFN can be reversed by the ceramide antagonist S-1-P. Inhibition of CFU-E colony formation by ceramide and by are both reversed by high concentrations of rhEPO. These findings strongly suggest that ceramide mediates inhibition of human CFU-E colony formation by gammaIFN.     

Adaptation of mouse leukemia cells L1210 to vincristine. Evidence for expression of P-glycoprotein.

A vincristine resistant cell line was obtained from mouse leukemia cells L1210 by long-term adaptation in a medium with stepwise increasing concentrations of vincristine. By Western blotting using monoclonal antibody C219, positive signal on the presence of P-glycoprotein was observed in the resistant cells. Moreover, hybridization of mRNA from vincristine resistant cells with radiolabeled MDR1 cDNA probe gave evidence about the expression of MDR1 gene. The observed resistance may be depressed by application of "chemosensitizers" such as (1) calcium entry blockers (verapamil and nifedipine); (2) neuroleptics (trifluorperasine) and (3) local anesthetics (lidocaine) directly to the grow medium. Any significant effect in O2 consumption as well as incorporation of [U-14C]-glucose by the sensitive or resistant cells was not detected in the absence of vincristine. Presence of vincristine induced increasing velocity of O2 consumption by resistant cells from 2.5 +/- 0.3 to 3.3 +/- 0.2 microliters/min.10(6) cells, and, on the other hand, decreasing O2 consumption by sensitive cells from 2.3 +/- 0.2 to 1.7 +/- 0.1 ml/min.10(6) cells. The presence of vincristine induced less potent decrease in glucose incorporation by resistant cells in comparison with values which were observed in sensitive cells.     

The chromosomes of L1210 mouse leukemia cells: G-banding, C-banding, and Ag-staining

The karyotype of L1210 mouse leukemia cells was studied using G-banding, C-banding and Ag-staining methods. In modal chromosome number and G-banding patterns we were interested in the differences between line A and B carried by DBA/2 mice in our laboratory. Both lines had the modal chromosome number of 38. Lines differed significantly in the average number of nine chromosomes per karyotype, whereas in marker chromosomes significant difference was found only in M2 and M6. Marker M1 t(5, 12) was present in 95% of cells in line A and 100% of cells in line B. More than 50% of cells in both lines contained M2, M11, M13 and M14. X-Chromosome and chromosome No. 4 were most frequently absent. More than 80% of cells in both lines had only one chromosome No. 5, and trisomy of chromosome No. 15 was found in 50% of cells in line A and 40% in line B. Values for disomy were low for all chromosomes in both lines. Marker M1 t(5, 12) possessed C-banding material at the proximal end of translocated chromosome No. 5 and NOR at the proximal end of translocated chromosome No. 12. Marker M2 t(1, 12) without NOR had the C-banding material located at the proximal end of chromosome No. 1.     

Isolation of a colony-stimulating factor produced by L 1210 murine leukemia cells

Summary Murine L 1210 leukemia cells spontaneously produce very low amounts of colony stimulating factor (CSF). CSF production was markedly increased by stimulating L 1210 cells with lipopolysaccharide, lectins, and sheep red blood cells. From the conditioned medium of phytohemoagglutinin-stimulated L 1210 cells we isolated a CSF with an apparent molecular weight of approximately 27,000. This CSF promoted the proliferation and the differentiation of murine GM-CFU showing a weak differentiation-inducing activity on WEHI-3 D (+) cells.     

Synergy between low-dose chemotherapy and immunotherapy in mouse L1210 leukemia

A high dose of 4 mg/kg of daunorubicin (DAU) given in combination with immunostimulation by the P40 immunomodulatory fraction of Corynebacterium granulosum and glutaraldehyde (GA)-treated tumor cells coupled with tetanus toxoid (P40 + GA-L1210-Tet) was more effective than DAU alone for treatment of L1210 mouse leukemia. A combination of low doses of DAU (0.0625-0.25 mg/kg) and P40 + GA-L1210-Tet was more effective than either P40 + GA-L1210-Tet or DAU alone. The cured mice were resistant to challenge with a high tumorigenic dose of L1210 tumor cells. A combination of various doses of mitomycin (1-4 mg/kg) with P40 + GA-L1210-Tet was more effective than mitomycin alone and was at least as effective as P40 + GA-L1210-Tet alone. Administration of DAU (0.5-4 mg/kg) to noninoculated C57BL/6 X DBA/2 mice resulted in increase of stimulation in vitro of collected spleen cells by mitogens.     

Endogenous erythroid colony formation by peripheral blood mononuclear cells from patients with myelofibrosis and polycythemia vera.

Peripheral blood mononuclear cells from patients with polycythemia vera or myelofibrosis with myeloid metaplasia were studied for their erythroid colony growth characteristics in plasma clot cultures. In both diseases, erythroid colonies formed early in culture in the absence of added erythropoietin (endogenous colonies). In no instance did early, endogenous colony formation occur with peripheral blood cells from normals or patients with secondary polycythemia. A normal response to erythropoietin was observed with both control and patients' peripheral blood cells. Spleen mononuclear cells obtained from one patient with myelofibrosis also produced endogenous colonies and showed a response to erythropoietin. This study suggests that culture of peripheral blood mononuclear cells might serve as a useful tool in discriminating polycythemia vera from secondary polycythemia.     

Separation and properties of DNA polymerase from murine leukemia L1210 cells.

The possible existence of several species of DNA-dependent DNA polymerases in mammalian cells in addition to those 2 polymerases which are the smaller enzyme from nucleus and larger one from cytoplasm each having distinct characteristics, have been reported recently. In order to examine the heterogeneity of DNA polymerases in murine leukemia L1210 cells and to characterize their general properties, we have attempted to separate the DNA polymerase activities from L1210 cells. By diethylaminoethyl (DEAE)-cellulose chromatography (0.2 M-1M KCl) of the whole cell extract from L1210 solubilized by 1% Triton X-100 and 0.5 mM ethylenediaminetetraacetate (EDTA), 4 fractions with DNA-dependent DNA polymerase activities were obtained and designated as DD-1, DD-2, DD-3, and DD-4 for eluents with each corresponding concentration of 0.2, 0.3, 0.5, and 0.7 M KCl, respectively. They were distinguishable in properties such as template preference, divalent cation requirement, DNase sensitivity, isoelectric point (pI) and the behavior on the phosphocellulose chromatography. DD-1 preferred native DNA as template exhibiting similar characteristic as nuclear polymerase with low molecular weight and insensitivity to SH-inhibitors. DD-2, DD-3, and DD-4 utilized activated DNA most efficiently, while activity of DD-3 increased even in the presence of DNase 1 under the condition where the others were completely inhibited. Distribution of DNA polymerase activities in the cells is discussed briefly.     

Mechanism of action of 1-beta-D-arabinofuranosyl-5-azacytosine and its effects in L1210 mouse leukemia cells

Ara-5AC depresses the growth of L1210 cells in vivo in a manner that is schedule-independent and at the dose levels which are similar to those of ara-C. The 50% inhibitory concentration for ara-5AC in L1210 system is about 0.75 microM. In distinction to ara-C the drug does not elicit the proliferation of proerythroblasts in the mouse bone marrow. It is phosphorylated by dCyd kinase, and the respective Km value is 70 microM. Ara-5AC is incorporated into DNA and almost completely blocks the incorporation of thymidine at a concentration of 10 microM.     

Steroid receptors and steroid response in cultured L1210 murine leukemia cells.

Murine L1210 leukemia cells were cultured in the presence of various steroid hormones to determine their growth response. Cells maintained in medium containing glucocorticoids such as dexamethasone exhibit a 10--20% inhibition of growth, whereas progesterone of 17 beta estradiol produce a 40--50% inhibition of growth. The response is due to growth inhibition and not to any cytolytic effect of the steroids. L1210 cytoplasmic and nuclear extracts contain high affinity binding sites for [3H]dexamethasone, [3H]progesterone and [3H]17 beta-estradiol. Competition with various steroids indicate that estrogens bind to one class of cytoplasmic binding sites, while glucocorticoids and progestins bind to a second class of receptor sites. These receptors appear not be be completely responsible for growth inhibition since there is no correlation between the magnitude of growth inhibition and the intracellular concentration of receptor sites, and since the concentration of steroid required to cause significant growth inhibition exceeds those concentrations required to saturate the cytoplasmic receptors.     

Insulin stimulates erythroid colony formation independently of erythropoietin

Summary . The effect of insulin on the proliferation of late stage erythroid precursor cells (CFU-e) from fetal mouse liver and aduit bone marrow was studied in a serum-free culture system. Insulin in supraphysiological concentrations (> 10 ^?8m ) stimulated erythroid colony formation independently of Ep. The combined effect of Ep and insulin was smaller than the surn of their single effects. The number of colonies obtained with insulin was linearly related to the number of plated cells. These results suggest that insulin stimulates erythroid colony formation by a direct action on CFU-e.     

Endogenous erythroid colony formation in myeloproliferative diseases does not depend on T cells

We investigated the influence of T-cell depletion on BFU-E and CFU-GM colony growth in vitro from normal individuals and patients with chronic granulocytic leukemia (CGL), idiopathic myelofibrosis (MF), and polycythemia vera (PV). Preincubation of mononuclear cells with the complement-fixing monoclonal antibody OKT11A, which is cytotoxic to T-lymphocytes, significantly reduced the number of erythropoietin (epo)-dependent BFU-E colonies cultured from normal bone marrow and normal peripheral blood, as well as from the blood of patients with CGL, PV, and MF. In contrast, the numbers of epo-independent ("endogenous") BFU-E colonies cultured from the blood of PV and MF patients were the same before and after T-cell depletion. The blood and marrow of CGL patients and normal individuals produced no epo-independent BFU-E proliferation. The growth of day-7 and day-14 CFU-GM was not significantly influenced by T-cell depletion in the majority of experiments. We conclude that T cells promote the growth of epo-dependent BFU-E colonies in vitro, but they do not influence the growth of "endogenous" BFU-E colonies from patients with myeloproliferative disorders.     

In vitro erythropoietin assay based on erythroid colony formation in fetal mouse liver cell culture

Critical studies were made on erythroid colony formation from cultured fetal mouse liver cells in an attempt to develop a simple and sensitive erythropoietin (Epo) assay procedure. The maximum colony formation was observed 24 h after plating of the cells when an evident dose-response relation was found for Epo added. The colony forming ability decreased steadily as the gestational age of the fetus advanced and was gradually lost by postnatal days 10-11. By morphological and cytochemical criteria almost all the colonies were found to be erythroid. 59Fe-labelling experiments revealed a fairly good correlation between the colony number and 59Fe incorporation into both cells and haem. Dose-response curves for plasma were parallel to the Epo standard curve. Based on these findings we developed a procedure which could measure as little as 0.4 mU of Epo without requiring 59Fe. Using this method, plasma Epo titres were determined in 16 normal and 69 anaemic subjects.     

Cobalamin-dependent replication of L1210 leukemia cells and effects of cobalamin analogues

L1210 cell line cells were made deficient in Cbl by propagation in a medium devoid of CNCbl in which fetal calf serum had been replaced by bovine serum albumin. These Cbl-deficient cells gradually ceased to multiply when the medium contained 5-CH3THF, although cell growth was resumed following the addition of CNCbl, OHCbl or folic acid. The results of this study provide experimental proof for the 'methyl trap' hypothesis. In contrast to the above effect of CNCbl, cobinamide and Cbl analogues which were produced by a reaction of OHCbl with ascorbic acid did not have any growth-inducing effect on the cells which had been cultured in a 5-CH3THF-supplemented medium and had ceased to multiply. Nor did these analogues have an inhibitory effect on CNCbl-dependent growth.     

The relationship of hemoglobin synthesis to erythroid colony and burst formation.

We have demonstrated that the cyclohexanone method for the extraction of hematin can be used to measure hemoglobin synthesis induced by erythropoietin (epo) in mouse bone marrow cells cultured in medium containing methyl cellulose. The time course of hemoglobin synthesis by mouse marrow cells showed two effects due to epo: an increase in hemoglobin synthesis at day 2, which corresponded to the formation of small erythroid colonies resulting from the CFU-E (colony-forming unit, erythroid), and a very large increase in hemoglobin synthesis, which was maximal at days 7-8 and corresponded to the formation of large erythroid colonies (bursts) resulting from the BFU-E (burst-forming unit, erythroid). The epo dose-response curves for CFU-E colony counts and day-2 hemoglobin synthesis were similar, and the cell-number-response curves for these two paramaters were parallel. The epo dose-response curve for BFU-E colony counts reached a plateau at an epo concentration between 3 and 5 units/ml, whereas the dose-response curve for 6-8-day hemoglobin synthesis did not reach a plateau even at an epo dose of 10 units/ml.     

Inhibition by mouse serum of hemopoietic colony formation in vitro.

The inhibitory activity of BALB/c serum for granulocytic and macrophage colony formation by mouse marrow cells in vitro was separable by flotation centrifugation into the very light density (VLD) fraction. On gel filtration the inhibitory material had an apparent molecular weight of 250,000 and on electrophoresis was localized as a single peak near the albumin region. These inhibitory preparations exhibited lack of specificity and inhibited colony formation by normal B-lymphocytes, lymphoid leukemic, plasma cytoma and mastocytoma cells. Preincubation of C57BL marrow cells at 37 degrees C with high concentrations of VLD fractions was cytotoxic and completely suppressed colony formation. Incubation at 0 degrees C or at 37 degrees C with low VLD concentrations caused a pattern of colony formation similar to that observed in cultures of marrow cells from mice with high serum inhibitory activity (CBA, BALC/c). The lack of specificity of serum inhibitors casts doubt on the role of these inhibitors as specific in vivo regulators of granulopoiesis and monocyte-macrophage formation.     

The cytostatic effects and mechanism of action of antiviral acyclic adenine nucleotide analogs in L1210 mouse leukemia cells

The growth and DNA synthesis of mouse leukemic cells L1210 in vitro was inhibited by (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA) by 50% at concentrations of 57.0 and 185.0 mumols/l, respectively, 9-(2-Phosphonylmethoxyethyl)adenine (PMEA) inhibited the cell growth and DNA synthesis at concentrations of 15.5 and 250.0 mumols/l. The 2-amino congeners of the above analogs were still more efficient: The corresponding values for 2-amino-PMEA were 6.0 and 10.0 mumols/l, and for 2-amino-HPMPA 19.5 and 50.0 mumols/l, respectively. The results suggest that probably at least a part of the growth inhibitory action of PMEA is due to mechanisms not related to its interference with the DNA synthesis.     

Spontaneous erythroid colony formation in Brazilian patients with sickle cell disease.

The ability of circulating progenitor cells to develop erythroid colonies was studied in vitro in the presence or absence of growth factors (5637-CM and erythropoietin) in 63 patients with sickle cell disease (SCD) (36 homozygotes for hemoglobin [Hb] S, 13 double heterozygotes for Hb S and beta thalassemia, and 14 SC patients) in Southeast Brazil. In the presence of growth factors, SCD patients (all genotypes) presented significantly higher numbers of circulating burst-forming unit-erythroid (BFU-E/5 x 10(5) MNC), when compared with control subjects. However, when the progenitor cells were cultured in the absence of added stimulus, high numbers of BFU-E were observed only in the genotypes SS and S/beta thalassemia. SC patients presented a similar response to the control subjects. Moreover, there was an inverse correlation between spontaneous (without stimulus) BFU-E and Hb levels in SCD patients. These results suggest that the formation of spontaneous BFU-E observed in SCD may be due to an expanded erythropoiesis secondary to hemolysis.