Alterations of glycolipids of human leukemia cell line HL-60 during differentiation

Alterations of glycolipids of human leukemia cell line HL-60 during differentiation were investigated. The neutral glycolipids of HL-60 cells are of the lacto-types such as lactosylceramide (LacCer) and lactoneotetraglycosylceramide (nLcOse4Cer). The gangliosides of HL-60 cells consisted of a mixture of the lacto-types and the ganglio-types. Chemical inducers of differentiation, 12-O-tetradecanoylphorbol 13-acetate (TPA) and dimethyl sulfoxide, altered the glycolipid profile; II3-alpha-acetylneuraminyl-lactosylceramide (GM3) increased in amount and LacCer and nLcOse4Cer decreased. Tunicamycin had no effect on the glycolipid synthesis. The GM3 accumulated more than tenfold in the TPA-induced macrophage-like cells. This accumulation could be due to the increased activity of LacCer sialytransferase.     

Comparison of histone variant synthesis in human lymphocytic leukemia cells and in normal lymphocytes

The synthesis of core histone variants and of histone H1 variants was determined in fresh leukemic cells of eight patients with leukemia [seven acute lymphoblastic (ALL) and one chronic lymphocytic (CLL)], in normal lymphocytes from healthy donors or from ALL patients in complete remission. Histone variant synthesis was evaluated by incubating cells with [14C]Lys and [3H]Arg in medium without Lys and Arg and then by two-dimensional polyacrylamide gel electrophoretic separations (acetic acid-urea-Triton x-100 acetic acid-urea-hexadecyltrimethylammonium bromide for core histone variants; sodium dodecyl sulfate/acetic acid-urea-hexadecyltrimethyl ammonium bromide for H1 variants). As previously reported, quiescent lymphocytes and lymphocytes stimulated with phytohaemagglutinin (PHA) showed clearcut changes in the proportions of synthesis of core histone variants and H1 variants. Leukemic lymphocytes freshly obtained from blood showed a pattern of core histone synthesis and H1 synthesis intermediate between that of quiescent and PHA-stimulated lymphocytes; this is probably due to the presence of a mixture of resting and growing cells. When leukemic cells were stimulated to grow by mitogens, the pattern of core histone and H1 variant synthesis was similar to that in mitogen-stimulated normal lymphocytes. Histone variants whose synthesis is associated with the S-phase were not synthesized in leukemic cells treated with the DNA synthesis inhibitors hydroxyurea and 1-beta-D-arabinofuranosylcytosine (Ara-C). The pattern of acetylation of histone H4 was also apparently similar in leukemic cells and normal lymphocytes. The radioactivity associated with the ubiquitinated forms of H2A increased in nongrowing lymphocytes and in leukemic cells treated with DNA synthesis inhibitors whereas they decreased after mitogenic stimulation. Variability was wide in the synthesis of ubiquitinated H2A in different cases of leukemia. The only clear-cut difference between leukemic cells and normal lymphocytes was that leukemic cells from ALL patients, but not lymphocytes from normal donors or from ALL patients in complete remission, synthesized appreciable amounts of H1 degrees, increasing after hydroxyurea/Ara-C treatment and decreasing after PHA-stimulation. In leukemic cells from a CLL patient H1 degrees synthesis was undetectable.     

Inhibitory effects of physalin B and physalin F on various human leukemia cells in vitro.

Physalins B and F were isolated and characterized from the ethanolic extract of the whole plant of Physalis angulata L. (Solanaceae). Both physalin B and physalin F inhibited the growth of several human leukemia cells: K562 (erythroleukemia), APM1840 (acute T lymphoid leukemia), HL-60 (acute promyelocytic leukemia), KG-1 (acute myeloid leukemia), CTV1 (acute monocytic leukemia) and B cell (acute B lymphoid leukemia). Physalin F showed a stronger activity against these leukemia cells than physalin B, especially against acute myeloid leukemia (KG-1) and acute B lymphoid leukemia (B cell). From the structural features, the active site seems to be the functional epoxy group for physalin F and the double bond for physalin B located at carbon 5 and 6; the former is much more active than the latter as regards anti-leukemic effects.     

Response of human myeloid leukemia cells to various sources of colony-stimulating activity and phytohemagglutinin-conditioned medium

The response of human myeloid leukemia cells to various sources of colony-stimulating activity (CSA) and media conditioned by phytohemagglutinin-stimulated mononuclear cells (PHA-LCM) was investigated in liquid and colony culture. PHA-LCM, placenta-conditioned medium, GCT cell line-conditioned medium, leukocyte-conditioned medium, and partially purified CSA for human and murine cells were tested for ability to support growth of granulocyte-macrophage colonies from adherent cell-depleted human bone marrow. This activity was correlated with ability to support leukemia colony growth in methylcellulose, and [3H]thymidine incorporation in liquid culture by normal bone marrow cells, leukemia cells, and the KG-1 myeloid leukemia cell line. For normal cells, growth and liquid culture responses were highly correlated for various sources of CSA (r = 0.92), and addition of data using PHA-LCM changed results only slightly (r = 0.89). [3H]thymidine incorporation by leukemia cells from patients without a prior history of a myeloproliferative disorder was also highly correlated with normal CSA (r = 0.97) for sources other than PHA-LCM. Responses of leukemia blasts and KG-1 cells in liquid culture to PHA-LCM appeared in excess of its CSA for normal cells. Colony growth by leukemia cells was not clearly correlated with either liquid culture activity for leukemia cells or CSA for normal cells. PHA-LCM was also not statistically superior to placenta-conditioned medium as stimulus for leukemia colony growth, but was superior to placenta-conditioned medium for some patients. Differentiation in culture did not appear to depend on CSA source. We conclude that normal myeloid cells respond to CSA in a highly correlated fashion in both colony and liquid cultures. The majority of myeloid leukemia cells respond to either PHA-LCM or CSA, but the ability of PHA-LCM to support leukemia cell growth is greater than its CSA content. The possibility exists that overlapping populations responsive to CSA and to PHA-LCM are present simultaneously in patients with myeloid leukemia.     

Interactions of thrombospondin with sulfated glycolipids and proteoglycans of human melanoma cells

Human melanoma cell spreading on thrombospondin substrates and chemotaxis in a gradient of soluble thrombospondin requires the amino-terminal heparin/sulfatide-binding domain of thrombospondin. Some melanoma cell lines attach but do not spread or respond in chemotaxis assays. Sulfated glycoconjugates produced by melanoma cells that could mediate these activities were identified by metabolic labeling with [35S] sulfate and tested for their ability to bind thrombospondin. Heparan sulfate proteoglycans that bind thrombospondin are made by both spreading and non-spreading melanoma cell lines. Thrombospondin binds with high affinity to a high molecular weight heparan sulfate proteoglycan, but not to the major chondroitin sulfate. The active heparan sulfate proteoglycan can be partially purified by affinity chromatography on thrombospondin-agarose or hydrophobic interaction with octyl-Sepharose. Thrombospondin binding requires the amino-terminal domain and is inhibited by monoclonal antibody A2.5 or fucoidan. Binding activity is lost following degradation of the proteoglycan with heparatinase or nitrous acid. [35S]Sulfate labels several melanoma cell glycolipids including galactosylceramide-I3-sulfate, lactosyl ceramide-II3-sulfate, and sulfated glucuronosylparagloboside. The latter glycolipid was detected in three cell lines that spread on thrombospondin but not in the nonspreading C32 melanoma cells. Thrombospondin binds to the isolated glycolipid, and the glycolipid and an antibody to this structure inhibit cell spreading on thrombospondin substrates. Thus, the presence of glycoconjugates with terminal nonreducing glucuronosyl 3-sulfate correlates with melanoma cell spreading on thrombospondin, whereas expression of heparan sulfate proteoglycans that bind thrombospondin does not.     

Etoposide-induced DNA cleavage in human leukemia cells

Summary The nuclear enzyme, topoisomerase II, is the major site of action for cancer chemotherapy agents such as etoposide, teniposide, and a variety of intercalating agents. These compounds cause the enzyme to cleave DNA, forming a DNA-protein complex that may be a key step leading to cell death. It is apparently unique as a chemotherapy target, since drug potency diminishes with decreasing enzyme activity. It was thus of interest to examine the topoisomerase content and drug-induced DNA cleavage in freshly obtained human leukemia cells and to compare the obtained data with the results of similar studies performed in well-characterized human leukemia cell lines. The human T-lymphoblast line, CCRF-CEM, was more than 100-fold more sensitive to the DNA-cleavage effect of etoposide than the cells of the 13 leukemic patients examined. One of the leukemia lines (HL-60) and a lymphoblastoid line (RPMI-7666) were somewhat less sensitive than cells of the CCRF-CEM cells, but were still 10-fold more sensitive than the patients studied. The relative insensitivity of the freshly obtained cells could not be accounted for by differences with respect to drug uptake but were associated with markedly reduced topoisomerase-II content as assayed by immunoblotting using a mouse polyclonal serum against topoisomerase II. Heterogeneity was observed in the sensitivities of patients' cells with respect to both drug-induced DNA cleavage and enzyme content. The observed differences between cultured cell lines and patients' cells may have been related to their proliferative status. Etoposide potency in normal resting lymphocytes resembles that observed in circulating leukemia cells. However, following mitogenesis with phytohemagglutinin and interleukin-2, proliferating lymphocytes become as sensitive to etoposide as cultured cell lines with regard to DNA cleavage. This effect was accompanied by an increase in topoisomerase-II content. Our data thus support the hypothesis that topoisomerase-II content may be an important determinant of cell sensitivity to certain classes of chemotherapy agents. Efforts to stimulate topoisomerase-II content may improve the therapeutic efficacy of these drugs.This work was supported by a USPHS grant (CA 40884)     

Adriamycin-induced DNA damage in human leukemia cells

The ability of adriamycin to alter integrity of DNA in leukemia cells from patients with acute lymphocytic leukemia, acute myelocytic leukemia, and chronic lymphocytic leukemia was tested. DNA damage (i.e. alkaline lability or strand scission) was found in most cell samples after a 2 h incubation with adriamycin (1.0 μg/ml). In cultured human leukemia cells (CCRF-CEM) DNA damage was progressive, continuing with no evidence of DNA repair after removal of adriamycin from the growth media. Analysis of human leukemia cells taken from patients before and during adriamycin therapy revealed extensive drug-induced DNA strand breakage, consistent with in vitro observations.     

Increased content of calmodulin in human leukemia cells

The content of calmodulin, as measured by a radioimmunoassay, in homogenates of two human leukemic cell lines (IM9 and Molt-4) was about 10-fold higher than that of normal human peripheral lymphocytes. These elevated calmodulin levels existed regardless of the proliferation status of the cells. Normal human lymphocytes stimulated with concanavalin A (con A) did not exhibit these elevated calmodulin levels. Likewise, leukemic cells which were ‘blocked’ from dividing through the addition of thymidine did not exhibit lower or ‘normal’ cellular calmodulin levels. These increased levels of calmodulin could contribute to the altered calcium regulation which exists in human leukemic lymphocytes.     

Binding of adult T-cell leukemia virus to various hematopoietic cells

A newly found human retrovirus, adult T-cell leukemia virus (ATLV) was shown by means of membrane immunofluorescence to bind to various hematopoietic cells including T-, B- and non-T, non-B-cell lines. Partially purified viral gp46 from culture fluids of ATL virus producer lines also bound efficiently to an ATLV-negative T-cell line, CCRF-CEM cells. When the viruses were pre-incubated with anti-ATLV-positive human sera, ATLV binding to the cells was clearly inhibited but not by pre-incubation with anti-ATLV-negative sera. These data suggest that: (1) ATLV binds not only to T-cells but also to multiple types of cells of hematopoietic origin; (2) anti-ATLV antibody-positive human sera have the blocking antibody for the binding of ATLV to lymphoid cells.     

MZ3 induces apoptosis in human leukemia cells

Purpose 4-(4-Bromophenyl)-2,3-dihydro-N,3-bis(3,4,5-trimethoxyphenyl)-2-oxoidmi-dazole-1-carboxamide (MZ3) is one of the synthesized combretastatin-A-4 analogues and has been reported that it displayed a promising specific activity against leukemia cell lines. Our purpose was to investigate the mechanism of MZ3’s cytotoxicity. Methods Cytotoxicity was measured by MTT method, apoptosis was measured by flow cytometry. DNA fragmentation was tested by agarose gel electrophoresis. Mitochondrial membrane potential (ΔΨm) was detected by JC1 staining and flow cytometry, while intracellular reactive oxygen species (ROS) was detected by 5-(and-6)-carboxy-2′-7′-dichlorofluorescin diacetate staining and flow cytometry. Protein expression was analyzed by western blotting. In vivo activity of MZ3 was assayed through severe combined immunodeficiency (SCID) mice model of human leukemia engrafts. Results MZ3 exhibited high anti-cancer activity in six leukemia cell lines, including two drug-resistant cell lines. MZ3 induced DNA fragmentation, and caused an elevation of ROS and a loss of ΔΨm in HL60 cells. MZ3 also induced the activation of caspase-3, influenced the expression of Bcl-2 family members, MAPKs and other proteins relative to mitochondria-induced apoptosis. In addition, N-acetylcysteine cannot inhibit HL60 cell apoptosis caused by MZ3. Furthermore, a prolonged survival time was observed after treatment with MZ3 in SCID mice model of human leukemia engrafts. Conclusions MZ3 is a potent compound against leukemia cell lines both in vitro and in vivo, and the mitochondrial pathway mediated by Bcl-2 protein family and MAPKs might be involved in signaling MZ3-induced apoptosis.     

Expression of glycolipids bearing Lewis phenotypes in tissues and cultured cells of human gynecological cancers.

Transformation-associated expression of Le(b) (Lewis antigen-b) or Le(Y) in human colorectal carcinomas has been well described. To examine the expression of glycosphingolipids (GSLs) bearing Lewis-phenotypes in human gynecological carcinoma-derived cells, we determined the concentrations of all GSLs. Although neither Le(b) nor Le(Y) was present in HEC-108 cells established from the poorly differentiated type of endometrial adenocarcinoma, other cell lines from moderately or well-differentiated types expressed either Le(b) or Le(Y), or both, at concentrations of 0.01 to 0.03 microg per mg of dry cells, which comprised 0.3 to 1.3% of the total GSLs. In the cervical and ovarian carcinoma-derived cell lines, Lewis phenotypes tended to be carried by nLc(4)Cer, which was accumulated in the cells without sialylation or fucosylation. These results indicated that expression of Le(b)- or Le(Y)-phenotypes was strongly dependent on the metabolic ability to supply the precursor GSLs. Both Le(b) and Le(Y) were successfully detected by monoclonal antibody MSN-1, which was a useful probe for the simultaneous detection of Le(b) and Le(Y). On application of MSN-1, either Le(b) or Le(Y) was detected in tissues from patients with well- and moderately differentiated types of endometrial adenocarcinoma at concentrations of 0.01 to 0.04 microg per mg of dry tissues, but not in the tissues of poorly differentiated type. Normal endometria at the follicular and luteal phases also contained the antigens, but the concentrations and the frequency of antigen expression were lower than those in the well- and moderately differentiated types of endometrial adenocarcinoma.     

Ether-linked phosphoglyceride content of human leukemia cells

The glycerolipids of most cells are characterized by a specific proportion of ether linkages at the sn-1 position of the glycerol backbone. A number of tumors are known to have altered concentrations of ether-linked lipids compared to normal tissues. However, no through examination of the ether-lipid content of human leukemia cells has been reported despite the importance of these lipids in normal leukocyte function. In the present study samples were obtained from adults with acute myelogenous leukemia (AML), chronic granulocytic leukemia in blast crisis, and acute lymphocytic leukemia and from healthy human donors. The cellular lipids were extracted, the individual phospholipid classes were isolated, lipid phosphorus content was determined, and the lipids were converted to diglyceride benzoate derivatives for separation and quantitation of the subclasses by high performance liquid chromatography. The data indicate that all the leukemic cells analyzed have an altered phospholipid composition compared to their respective normal leukocytes. Furthermore, among the AML patients both the percentage of the choline-containing phosphoglyceride fraction (PC) which is alkyl linked and the nmoles alkyl-PC/10(6) cells differ significantly by FAB subtype. A positive correlation between the levels of alkyl-PC and the degree of cellular differentiation is observed. Although no differences are observed between chronic granulocytic leukemia in blast crisis and AML lipids, the leukemic cells contain dramatically lower levels of alkyl-linked PC than do normal polymorphonuclear leukocytes. In contrast, no differences are observed between the alkyl-PC content of normal and leukemic lymphocytes. In light of the relations among ether-lipids, protein kinase C, and cell differentiation, these data suggest the ether-linked lipids are important in myeloid cell function and differentiation.     

Dietary bioflavonoids induce apoptosis in human leukemia cells

Dietary bioflavonoids are secondary metabolites of plants that are known to have a variety of bio-effects, including anti-cancer activity. In this study, we examined the effects of flavonoids on the growth of human leukemia cells and found that certain flavonoids induce apoptosis in a variety of human leukemia cells. The apoptosis induced by bioflavonoids was dose-dependent and was accompanied by a disruption of the mitochondrial transmembrane potential and the activation of caspase. Our data suggests that dietary bioflavonoids may be useful chemotherapeutic reagents for leukemia patients.     

Effects of various cytokines on proliferation of acute lymphoblastic leukemia cells

We investigated the effects of recombinant human interleukins 1 to 6 (rIL-1 to -6) on the proliferation of blast cells from patients with acute lymphoblastic leukemia (ALL). The 3H-TdR incorporation in the presence of various cytokines was examined in cells from 14 patients: 12 with B-lineage ALL, 1 with T-lineage ALL and 1 with biphenotypic leukemia. In B-lineage ALL, a significant increase in 3H-TdR incorporation was observed in 5/12 cases (42%) in the presence of rIL-1 alpha, in 10/12 cases (83%) with rIL-2, in 9/12 cases (75%) with rIL-3, in 3/6 cases (50%) with rIL-4, in 4/6 cases (67%) with rIL-5, and in 4/12 cases (33%) with rIL-6. The mean stimulation index of the cells showing a positive response was 1.74 for rIL-1 alpha, 3.40 for rIL-2, 2.55 for rIL-3, 1.86 for IL-4, 1.56 for rIL-5, and 2.97 for rIL-6. T-lineage ALL cells were stimulated only in the presence of rIL-2, and biphenotypic leukemia cells were not stimulated by any of the cytokines tested.     

Development of polyploidization in taxol-resistant human leukemia cells in vitro

The effects of taxol on antitubulin immunofluorescent staining patterns, cellular DNA content, and labeling with [3H]thymidine were measured for the taxol-sensitive HL60 and taxol-resistant K562 cell lines after exposures for 0, 4, 12, and 24 h. Taxol caused a relative increase in the fraction of 4C interphase and metaphase cells in both lines although the 4C interphase accumulation was greater for the resistant K562 line. Of the cells with S-phase DNA content, taxol-treated HL60 cells were less likely to incorporate [3H]thymidine than taxol-treated K562 cells. However, a decrease in percentage of S-phase labeling for both lines relative to control cells was seen. Finally, taxol induced the development of polyploid cells (cells with DNA contents greater than that of the 4C G2-M peak) in the relatively taxol-resistant K562 cells, an effect not seen in the relatively taxol-sensitive HL60 line. After 24 h of taxol exposure 70% of all K562 cells were polyploid while only 8% of the HL60 cells were polyploid. The capacity of K562 cells to generate polyploidy in response to taxol correlated with taxol resistance by previous assay and may be a useful indicator of drug resistance.     

不同白血病细胞对同器官侵袭能力的比较观察

Using patho-morphological method and transplantation bio-assay, the in vivo invasiveness of leukemia cells in three transplantable mouse T cell leukemia models was comparatively studied. The results showed that the invasion to the liver was consistent, but that to other organs was obviously different. L615 and L7212 leukemia cells preferred bone marrow and spleen to peritoneum but L7811 leukemia cells were just the opposite. Transplantation bio-assay demonstrated that leukemia cells were present in the bone marrow of L615 mice as early as 6 hours after leukemic cell inoculation, but no leukemia cells were detected in the bone marrow of L7811 mice 2 days after inoculation. In the terminal phase, L615 mice bone marrow became filled with leukemia cells, but L7811 mice bone marrow contained only a few leukemia cells. The difference in invasiveness of leukemia cells into organs is probably related to "homing" receptor. The same type of leukemia cells may possess multiple "homing" receptors.     

DNA damage by anthracycline drugs in human leukemia cells

Leukemia cells from 4 acute myelocytic leukemia (AML) and 1 acute lymphocytic leukemia (ALL) patients were incubated with a set of 6 anthracycline agents: Adriamycin (Am), 4′-epi-Adriamycin (4′-epi-Am), daunorubicin (Dm), 4-demethyoxy-daunorubicin (4-dDm), carminomycin (Cm) and N-trifluoroacetyl-Am-14-valerate (AD32). Cells were assayed for drug uptake after incubation for 2 h, and for DNA damage and drug retention 4 h later. Uptake and retention patterns were characteristic for each agent and fairly uniform for the different cell populations. In contrast, profiles of the amount of DNA damage produced reflected striking differences in each population of cells. These individual responses raise the possibility that leukemic cells resistant to one anthracycline may yet be sensitive to another.