Membrane potential in human myeloid leukemia cell line ML-1: responsiveness of granulocytic and monocytic differentiated cells.

The membrane potential responsiveness of human myeloid leukemia cells (ML-1 line) was studied with the voltage sensitive fluorescent dye diS-C3-(5). The experimental procedure used in this study enabled us to assess the magnitude of the membrane potential change in cells treated with ouabain, 12-0-tetradecanoylphorbol-13-acetate (TPA) and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), relative to the membrane potential in the untreated control. Inhibition of the Na, K-ATPase by ouabain was followed by a (20 +/- 4) mV depolarization. In undifferentiated homogeneous cell population TPA caused a (19.4 +/- 4.4) mV depolarization while FMLP had virtually no effect. Cells in which granulocytic or monocytic differentiation was induced by retinoic acid or 1,25-dihydroxyvitamin D3 exhibited under the effect of TPA a (57.8 +/- 7.1) mV and (34.8 +/- 10.9) mV depolarization, respectively. A very small transient depolarization was also observed up on treating of the cells with FMLP. The changes in the membrane potential responsiveness in the induced cells are obviously connected with the cell differentiation.     

Identification of transferrin as a progression factor for ML-1 human myeloblastic leukemia cell differentiation

We have previously demonstrated (Guan X.-P., Hromchak, R. A., and Bloch, A. (1989) Cancer Commun. 1, 111-115) that ML-1 human myeloblastic leukemia cells differentiate to monocyte/macrophage-like cells by the sequential action of competence and progression factors. Tumor necrosis factor-alpha, transforming growth factor-beta, and the phorbol ester tetradecanoylphorbol acetate were found to induce competence, whereas a 77-kDa glycoprotein (DF77) isolated from mitogen-stimulated human leukocyte-conditioned medium initiated progression. In this communication we show DF77 to be an isoform of human transferrin. Hemin or soluble iron complexes did not induce differentiation progression, suggesting that the participation of transferrin in ML-1 cell differentiation may not be related to its iron-carrying capacity.     

Synergism of leukemic blast growth factors in medium conditioned by human bladder carcinoma cell line 5637

Leukemic blast growth factors (LBGFs) are necessary for in vitro growth of clonogenic cells from patients with acute myeloblastic leukemia. As the human bladder carcinoma cell line 5637 had previously been reported to secrete abundant LBGFs into the culture supernatant, the LBGFs in 5637-conditioned medium (5637-CM) were characterized. Measurement of LBGFs was done using an in vitro leukemic blast colony assay in methylcellulose culture. LBGFs in 5637-CM were fractionated by anion exchange chromatography, and two peaks of activity were recovered. Pool B (high-salt eluent) and/or purified granulocyte colony-stimulating factor (G-CSF) were added to the clonogenic leukemic blast cell assays. It was found that pool B was more active than G-CSF in the majority of cases examined and that the two types of activity were synergistic in some cases.     

Functional changes in human leukemic cell line HL-60. A model for myeloid differentiation

Polar solvents induce terminal differentiation in the human promyelocytic leukemia cell line HL-60. The present studies describe the functional changes that accompany the morphologic progression from promyelocytes to bands and poly-morphonuclear leukocytes (PMN) over 9 d of culture in 1.3 percent dimethylsulfoxide (DMSO). As the HL-60 cells mature, the rate of O(2-) production increase 18-fold, with a progressive shortening of the lag time required for activation. Hexosemonophosphate shunt activity rises concomitantly. Ingestin of paraffin oil droplets opsonized with complement or Ig increases 10-fold over 9 d in DMSO. Latex ingestion per cell by each morphologic type does not change significantly, but total latex ingestion by groups of cells increases with the rise in the proportion of mature cells with greater ingestion capacities. Degranulation, as measured by release of β-glucuronidase, lysozyme, and peroxidase, reaches maximum after 3-6 d in DMSO, then declines. HL-60 cells contain no detectable lactoferrin, suggesting that their secondary granules are absent or defective. However, they kill staphylococci by day 6 in DMSO. Morphologically immature cells (days 1-3 in DMSO) are capable of O(2-) generation, hexosemonophosphate shunt activity, ingestion, degranulation, and bacterial killing. Maximal performance of each function by cells incubated in DMSO for longer periods of time is 50-100 percent that of normal PMN. DMSO- induced differentiation of HL-60 cells is a promising model for myeloid development.     

The role of interferon-gamma in induction of differentiation of human myeloid leukemia cell lines, ML-1 and HL-60

Highly purified natural interferon-gamma (IFN-gamma) induced differentiation having characteristics that are associated with the human promyelocytic leukemia cell line, HL-60. Monoclonal antibody to INF-gamma neutralized its activity. However, the natural IFN-gamma had almost no inducing activity in ML-1, a human myeloblastic leukemia cell line. Similar results were obtained using recombinant IFN-gamma. Mitogen stimulated human leukocyte conditioned medium (LCM) induced differentiation of both ML-1 and HL-60 cells. After treatment of LCM with monoclonal antibody to IFN-gamma, LCM activity was reduced more than 50% in ML-1 cells, and 80% in HL-60 cells. Even if IFN-gamma was eliminated from LCM by affinity chromatography, the LCM induced differentiation of ML-1 and HL-60 cells, but IFN-gamma markedly enhanced the ML-1 cell differentiation induced by IFN-gamma free LCM. The results suggest that leukocytes produce differentiation inducing factor(s) other than IFN-gamma, and that IFN-gamma is both an inducer and an enhancer of induction of human myelogenous leukemia cells.     

Retinoic acid acylation (retinoylation) of a nuclear protein in the human acute myeloid leukemia cell line HL60

all-trans-Retinoic acid is a potent inducer in vitro of the differentiation of the human acute myeloid leukemia cell line HL60 and of fresh cells from patients with acute promyelocytic leukemia. The recent discovery of nuclear retinoic acid receptors provides a basis for understanding how retinoic acid acts at the genetic level. We have now found that retinoic acid is incorporated into HL60 cells in a form that is not removed by extraction with CHCl3:CH3OH. About 90% of this labeled retinoic acid is trichloroacetic acid-soluble after digestion with proteinase K or after hydrolysis with either NH2OH or CH3OH:KOH under mild conditions. Methyl retinoate is the major product of hydrolysis with CH3OH:KOH. These results are consistent with retinoylation of protein with the formation of an ester, probably thioester, bond. The extent of the retinoylation of HL60 protein is dependent on both time and retinoic acid concentration. A major fraction of the retinoylation is of protein that has a molecular mass of 55 kDa after reduction with dithiothreitol. On two-dimensional gels, the retinoylated protein has a pI of about 4.9 and a molecular mass of 55-60 kDa. These characteristics and its localization in the cell nucleus are consistent with retinoylation of the HL60 nuclear retinoic acid receptor or a closely related protein.     

Dissociation of c-fos induction from macrophage differentiation in human myeloid leukemic cell lines.

Treatment of five human myeloid leukemic cell lines (KG1, ML3, HL-60, U-937, and HEL) with TPA was followed by macrophage differentiation and was accompanied by an early and transient increase in the mRNA level of c-fos proto-oncogene. The induction of c-fos was also observed in human cell lines K562 and K-Gla that did not respond to TPA with terminal macrophage differentiation. The treatment of HL-60 and U-937 cell lines with 1-oleoyl-2-acetylglycerol, a synthetic analog of diacylglycerol that, like TPA, stimulates protein kinase C activity, was followed by early and transient induction of c-fos mRNA in the absence of terminal macrophage differentiation. Finally, treatment of HL-60 with TPA in the presence of retinal, an inhibitor of protein kinase C, drastically reduced the induction of c-fos mRNA but had no effect on the terminal macrophage differentiation that is induced in this cell line by TPA. These results indicate that the induction of c-fos and terminal macrophage differentiation in response to TPA treatment can be dissociated in the in vitro models provided by human myeloid leukemic cell lines. Moreover, these findings suggest that the induction of c-fos is not only insufficient but may also be unnecessary for the differentiation along the monocyte-macrophage pathway.     

Maturation inducer activity and the process of human myeloid leukemia cell differentiation

The process and mechanism of human myeloid leukemia cell differentiation induced by T-cell lymphokine maturation inducer activity was investigated. The maturation inducer activity was purified from conditioned medium of normal peripheral blood lymphocytes and shown to be a 50,000 M.W. protein. The degree of maturation of myeloid cell cultures was directly related to the dosage of the inducer. The interaction of the leukemia cells with the inducer led to initiation of terminal differentiation to monocytic cells. Proliferation cessation of the leukemia cells and the expressions of mature monocytic cells indicated a continuous and multistaged process.     

Prostaglandin E2 regulates vitamin D receptor expression, vitamin D-24-hydroxylase activity and cell proliferation in an adherent human myeloid leukemia cell line (Ad-HL60).

The effects of prostaglandin E2, forskolin, and phorbol 12-myristate 13-acetate on cell proliferation, cell surface antigen expression, vitamin D-24-hydroxylase activity and vitamin D receptor (VDR) expression have been studied in an adherent variant (Ad-HL60) of the human HL60 promyelomonocytic leukemia cell line. Ad-HL60 cells have a more differentiated phenotype than the nonadherent HL60 cells from which they were derived and, unlike the parent cell line, constitutively express vitamin D-24-hydroxylase activity. Treatment of Ad-HL60 cells with 1 microM PGE2 resulted in a decrease in the rate of cell proliferation (cell numbers were approximately 23% of control values after 72 h treatment), a change in expression of leukocyte surface antigens (decreased CD13 and CD14, increased CD11b and CD49d expression), an increase in the synthesis of 24,25-dihydroxyvitamin D3 from substrate 25-hydroxyvitamin D3 (control 5.76 +/- 0.17, 72 h PGE2-treated cells 12.10 +/- 1.90 pmol/h/10(6) cells), and an increase in receptors for the active metabolite of vitamin D, 1 alpha,25-dihydroxyvitamin D3, from 3910 to 11285 receptors per cell in control and 7-day treated cells, respectively. Prostaglandin E2 may be acting via a mechanism involving cyclic AMP in these cells, as we have also demonstrated that 10 microM forskolin, an adenylate cyclase activator, has similar effects. Phorbol 12-myristate 13-acetate had little effect on any of the parameters measured in this cell line.     

Role of ROS and MAPK in TPA-induced ICAM-1 expression in the myeloid ML-1 cell line

Intercellular adhesion molecule 1 (ICAM-1) has been implicated in playing a key role in the mechanism of inflammatory process initiated in response to environmental agents, and during normal hematopoietic cell differentiation. Though induction of ICAM-1 by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in myeloid cells has been reported, the molecular mechanism by which TPA upregulates ICAM-1 expression remains unclear. In the present study, we investigated the signaling mechanism associated with TPA-induced ICAM-1 expression in ML-1 cells. Herein, our microarray, flow cytometry, and Western blot analysis indicated that ICAM-1 was constitutively expressed at a low level in ML-1 cells, but its expression was further upregulated at both the mRNA and protein levels in response to TPA. ICAM-1 expression in response to TPA was inhibited by pretreatment with GF109203X [a specific inhibitor of protein kinase C (PKC)], or with PD98059 and U0126 (specific inhibitors of MEK), suggesting the importance of PKC, and Erk1/2 signaling cascades in this response. Interestingly, ICAM-1 expression in response to TPA-induced PKC activation was linked to the generation of reactive oxygen species (ROS), as pretreatment with NAC (an ROS scavenger) blocked both ErK1/2 activation and ICAM-1 expression induced by TPA. In addition, TPA-induced ICAM-1 expression was blocked by inhibition of nuclear factor-kappaB (NF-kappaB) activation following pretreatment with BAY11-7085 (a specific inhibitor of NF-kappaB activation). TPA-induced NF-kappaB activation was shown by increased degradation of IkB (NF-kappaB specific inhibitory protein). Together, these observations demonstrated that TPA, a potent activator of PKC, induces ICAM-1 expression via a ROS- and ERK1/2-dependent signaling mechanism in ML-1 cells.     

Proteins in human myeloid leukemia cell line HL60 reacting with retinoic acid monoclonal antibodies

The vitamin A derivative, retinoic acid (RA) has various biological effects in mammalian cells and tissues. It is well known that RA induces differentiation of leukemia cells and inhibits cell growth. There are two pathways for RA action; one via RA nuclear receptors (RARs), and one via acylation of proteins by RA (retinoylation). However, an understanding of which actions of RA occur via RARs and which occur via retinoylation is lacking. Thus, we undertook the examination of HL60 proteins using anti-RA monoclonal antibodies (ARMAs). These ARMAs showed specific binding to proteins in a saturable manner depending on protein and antibody concentration. Proteins eluted by Mono Q anion exchange chromatography and separated using two-dimensional polyacrylamide gel electrophoresis were detected by ARMAs. One of these ARMA-bound proteins in HL60 cells was identified as alpha-actinin. These results indicate that retinoylated proteins in HL60 cells can be recognized by ARMAs and that alpha-actinin modified by RA may play a significant role in RA-induced differentiation, including the promotion of cytomorphology changes.     

Persistence of herpes simplex virus type-2 genome in a human leukemic cell line

To study the nature of virus-cell interaction in persistently infected cells we have examined production of infectious virus, synthesis of viral DNA and DNA polymerase in a human leukemic cell line K562. It was found that only one of three K562 cell lines was permissive for limited growth of HSV-2 and infectious virus was released in a cyclical fashion. Intranuclear inclusions with electron-dense fibrils and particles resembling viral structures were observed in the virus-infected but not control K562 cells. Viral DNA synthesis could not be detected by centrifugation in CsCl density gradients; but was readily identified by Southern blot hydridization of virus-infected intracellular DNA with purified viral DNA. Viral DNa polymerase was synthesized by infected cells during active infectious virus production. In one of the two K562 cell lines that did not produce infectious virus, a few DNA fragments from infected cells were found to hybridize with purified viral DNA. These results suggest that variable lengths of HSV-2 genome can be harbored and propagated by different human leukemic K562 cells.     

PKC inhibition is involved in trichosanthin-induced apoptosis in human chronic myeloid leukemia cell line K562

Trichosanthin (TCS), a type I ribosome-inactivating protein, induces cell death in various cell types including several tumor cell lines. However, the mechanism remains largely uncharacterized. In this study, we investigated the possible mechanism underlying its cytotoxicity by using human chronic myeloid leukemia cell line K562. We found that TCS induced apoptosis in K562 cells in a time- and concentration-dependent manner and can be blocked by caspase-3 inhibitors. Interestingly, TCS treatment induced a transient elevation in intracellular calcium concentration and a slow increase in reactive oxygen species production, while calcium chelators and antioxidants had no obvious effect on TCS-induced apoptosis, suggesting that calcium changes and reactive oxygen species may not be involved in TCS-mediated apoptosis in K562 cells. Instead we found that TCS partly inhibited PKC activity. Indeed, the PKC activator, PMA, inhibited while the PKC inhibitor, calphostin c, enhanced TCS-induced apoptosis. These PKC modulators had similar effects on TCS-induced cleavage of caspase-3, and caspase-3 inhibitors prevented calphostin c-enhanced apoptosis induced by TCS. In summary, we conclude that TCS induces apoptosis in K562 cells partly via PKC inhibition and caspase-3 activation.     

Interleukin 1 (IL 1)-dependent lymphokine production by human leukemic T cell line HSB.2 subclones

Cloning of a human T cell leukemic cell line, HSB.2, was performed by a limiting dilution method to obtain clones with high levels of IL 2 production. None of the subclones that were obtained produced IL 2 constitutively, and only a low level of IL 2 was produced by the stimulation of these subclones with phytohemagglutinin (PHA) alone. High levels of IL 2 production (greater than 300 U/ml) were observed in several clones when stimulated with a cocktail of PHA and IL 1. Among them, HSB.2-A7-D2, A7-D9, or C5-B2 subclones, which were selected after cloning twice, were most effective in IL 1-dependent IL 2 production. HSB.2 subclones exhibited IL 1-dependent production of a variety of lymphokines other than IL 2, e.g., interferon-gamma (IFN-gamma), B cell growth factor (BCGF), and colony-stimulating factor (CSF). We observed that subclones with high IL 2-producing capacity tended to produce high levels of IFN-gamma or BCGF as well, while the capacity of CSF production was not parallel to these properties. Although several subclones were found to produce IFN-gamma and BCGF simultaneously with minimal IL 2 activity, no subclones with an exclusive BCGF production were obtained. Furthermore, when supernatants from the stimulated A7-D9 subclone were applied to an Ultro-gel AcA54 gel chromatography, it was revealed that IL 2 activity (m.w. 17K to 18K) and IFN-gamma (40K to 45K) were clearly separated, whereas two peaks of BCGF activity coincided with each peak of IL 2 and IFN-gamma, respectively. On the other hand, CSF activity was eluted at a different peak (30K to 35K). These data indicate that IL 2, IFN-gamma, and CSF activities are based on distinct molecules, whereas BCGF activities are indistinguishable from IL 2 and IFN-gamma. The HSB.2 subclones thus selected will provide a useful model for delineating the mechanism of IL 1-dependent lymphokine(s) production, and are a promising candidate for better lymphokine(s) producers.     

PHA激活的脐血T细胞条件培养液对人骨髓CFU—C的抑制

Cord blood T cells were enriched by nylon wool colomn, and effects of PHA-stimulated T cell supernatant collected from 18 h to 7 days on the proliferation of CFU-c were studied. The results showed that the supernatant collected at 18 h (PHA-TCM) could significantly inhibit the growth of CFU-c and the inhibition was PHA-TCM dose dependent, suggesting there is a CFU-c inhibitory activity in PHA-TCM. Kinetic studies demonstrated that the activity was decreased in the supernatant collected at 48 h and disappeared at 7 days. On the other hand, unstimulated T cell supernatant and PHA alone had no inhibitory effect on CFU-c growth. Indomethacin did not affect the production of the inhibitory activity and no interferon activity could be detected in PHA-TCM. These suggested that the inhibition was mediated by a non-interferon, non-prostaglandin suppressor. Further studies revealed that the suppressor was a protein stable at 56 degrees C and lost in pH 2 and pH 11 for 3 h, its molecular weight was large than 10,000 dolton.     

Anti-MIF activity of serum from a rabbit immunized with human B-lymphoid cell line migration inhibitory factor.

Sensitized parental strain lymphocytes can specifically inhibit proliferation of semiallogeneic erythroid cells grafted to irradiated syngeneic F1 hybrid mice. In H-2-incompatible strain combinations, inhibition is mainly caused by a direct attack of the lymphocytes against the grafted bone marrow. However, some indirect effect is also caused by a reaction of the lymphocytes against cells of the recipient. In this investigation we have shown that lymphocytes which have been sensitized against Mls-antigen-incompatible cells inhibit erythroid proliferation indirectly by reacting against host cells. The sensitized lymphocytes exhibit very little direct effect on the grafted bone marrow cells.