Modulation of homeobox B6 and B9 genes expression in human leukemia cell lines during myelomonocytic differentiation

Homeobox genes (HOX) may have a regulatory function in the differentiation process of hematopoiesis. We examined the change of HOX B6 and HOX B9 mRNA expressions during the in vitro differentiation of four myeloid leukemia cell lines because HOX B6 may be involved closely in myeloid differentiation. HL-60, NB4, NKM-1 and NOMO-1 were established from acute leukemia of M2, M3, M2 and M5 subtype of the French-American-British classification, respectively. All-trans retinoic acid (ATRA), TPA, and G-CSF were used as differentiation inducers. Each cell line was cultured with each inducer and total RNA was isolated on day 1, 2, 3, or 5. HOX B mRNA was detected by Northern blotting and RT-PCR methods. HOX B6 and HOX B9 mRNAs were constitutively expressed in NB4, NKM-1 and NOMO-1, but were expressed at very low levels in HL-60. HOX B6 and HOX B9 mRNAs were also expressed in fresh acute myelocytic leukemia blasts. HOX B6 mRNA expression in HL-60, NB4, and NKM-1 cultured with ATRA increased on day 3 and decreased on day 5. HOX B6 mRNA expression in NB4 and NKM-1 cultured with TPA decreased on day 3. HOX B9 mRNA expression displayed changes similar to those of HOX B6 mRNA in NB4 and NKM-1. These results indicate that myeloid leukemia cell lines express HOX B6 and HOX B9, and that their respective mRNA expressions in NB4 and HL-60 increase at a mid stage of myeloid differentiation by ATRA induction and then decrease during a late stage. HOX B6 mRNA expression decreased in monocytoid differentiation by TPA induction in NB4, HL-60 and NKM-1. HOX B6 antisense-oligonucleotide inhibited the proliferation of NB4 and NKM-1. These results suggest that HOX B gene expression is related to simultaneous activation of cellular proliferation and differentiation in leukemic cells.     

Hydrocortisone inhibits granulocyte-macrophage colony-stimulating factor production from normal human peripheral blood mononuclear cells and CD3+ T cells

Normal human peripheral blood mononuclear cells (MNCs), particularly T lymphocytes (T cells), are a rich source of granulocyte-macrophage colony-stimulating factor (GM-CSF). Glucocorticoids are known to inhibit GM-CSF production in in vitro cultures of a human fibroblast cell line and in normal human blood monocytes and alveolar macrophages. To determine whether glucocorticoids also inhibit GM-CSF production from normal human MNCs and T cells, we set up cultures of normal human MNCs and T cells in a liquid system in the presence and absence of 5, 50, and 250 microg/dL of hydrocortisone, and an hour later, a constant dose of 50-ng/mL Escherichia coli lipopolysaccharide (LPS) or 10-microg/mL phytohemagglutinin (PHA) was added. After three days, cell counts and GM-CSF levels were determined. Administering 50- and 250-microg/dL hydrocortisone decreased lymphocyte recovery from MNC cultures with LPS (p < or = 0.01), and 250 microg/dL of hydrocortisone decreased lymphocyte recovery from MNC and T-cell cultures with PHA (p < or = 0.03). The amount of GM-CSF produced from PHA-stimulated MNCs was about 100-fold higher than that produced from LPS-stimulated MNCs. The magnitude of GM-CSFs produced in MNC and T-cell cultures stimulated by PHA was comparable (p=0.88). Administering hydrocortisone at 5, 50, and 250 pg/dL decreased GM-CSF production (p < 0.003) in LPS- or PHA-stimulated MNC cultures and in PHA-stimulated T-cell cultures. PHA (not tested with LPS)-stimulated GM-CSF messenger RNA (mRNA) expression was blocked by hydrocortisone. These results indicate that lower concentrations of hydrocortisone inhibit GM-CSF production from normal human blood MNCs and T cells entirely by inhibiting the expression of GM-CSF mRNA, and higher concentrations of hydrocortisone inhibit by a combined effect of inhibiting the expression of GM-CSF mRNA and decreasing the lymphocyte count.     

Increment of the cyclin D1 mRNA level in TPA-treated three human myeloid leukemia cell lines: HEL, CMK and HL-60 cells

To study the involvement of cyclins in cell-cycle progression, changes of mRNA levels for three G1 cyclins (cyclin C, D1 and E) and cyclin A were studied in a leukemia cell line, HEL cells, before and after incubation with 12-O-tetradecanoylphorbol-13-acetate (TPA). Unexpectedly, the cyclin D1 mRNA level markedly increased in the HEL cells when the cells were growth-arrested by TPA, while the amounts of cyclin E and A mRNAs decreased to an almost undetectable level in HEL cells after incubation with TPA. The similar marked increment of the cyclin D1 mRNA level was observed in other leukemia cell lines, CMK and HL-60 cells, after incubation with TPA. The cyclin C mRNA was not detected in HEL cells before and after incubation with TPA.     

Cell-water transfer and stability of biological structures (resonance of biological structures)

We demonstrated that adrenomedullin (AM) is produced and secreted from human leukemia cell lines (THP-1 and HL-60) as well as peripheral blood granulocytes, lymphocytes, monocytes and monocyte-derived macrophages. Immunoreactive AM accumulated in the culture media of THP-1 and HL-60 cells increased according to their differentiation into macrophage-like cells. Retinoic acid exerted synergistic effects on AM secretion from THP-1 and HL-60 cells when administered with tumor necrosis factor-alpha, lipopolysaccharide or 12-O-tetradecanoyl phorbol-13-acetate. AM was shown to increase the scavenger receptor activity on THP-1 cells. Thus, monocytes/macrophages should be recognized as sources of AM, and the secreted AM may modulate the function of macrophages.     

Western blotting analysis of heat shock proteins of Rickettsiales and other eubacteria

Promyelocytic leukemia HL-60 cells promoted by PMA to differentiate along the monocyte pathway adhere to tissue culture plates. To explore the regulation of adhesion molecules in cells promoted to differentiate, the expression and secretion of osteopontin (OPN) and expression of associated cell surface receptors, CD44 and integrin subunits alpha(v), beta3, beta1, were examined. Results were as follows: 1) PMA induced OPN mRNA and OPN secretion into media; 2) untreated cells expressed beta1 and CD44 mRNA, and PMA induced alpha(v), and beta3 mRNA and increased beta1 and CD44 mRNA expression; 3) PMA increased levels of alpha(v), beta3, beta1 and CD44 protein on the cell surface; and 4) retinoic acid, which promotes granulocytic differentiation of HL-60 cells, did not affect OPN, alpha(v), beta3, beta1, or CD44 mRNA or protein expression. These data suggest that induction of OPN and associated receptors may play a role during monocytic differentiation of HL-60 cells.     

Bronchial responsiveness to ultrasonic fog in occupational asthma due to toluene diisocyanate

Accumulating evidence suggests that prothymosin alpha has an as yet undefined intracellular, perhaps intranuclear, function related to cell proliferation. Prothymosin alpha mRNA and/or peptide levels increase when cells are stimulated to proliferate. Because proliferation and differentiation events are often inversely correlated, we examined prothymosin alpha gene expression during proliferation and differentiation of HL-60 myeloid leukemia cells. Steady-state levels of prothymosin alpha mRNA, which are high in exponentially growing HL-60, decrease within hours after induction of HL-60 to differentiate along the neutrophil pathway with dimethylsulfoxide (DMSO) or along the macrophage lineage with either tetradecanoylphorbol acetate (TPA) or bryostatin 1. The decline in prothymosin alpha mRNA in response to these differentiation signals parallels that of c-myc mRNA under the same conditions. We then determined whether the downregulation of prothymosin alpha and c-myc mRNA were due to differentiation or cessation or proliferation. Recombinant human gamma-interferon induces monocytic differentiation of HL-60, but permits continued proliferation, and, under these conditions, expression of prothymosin alpha, as well as of c-myc, mRNA remains elevated. We conclude that prothymosin alpha and c-myc expression are coregulated in differentiating HL-60 and that their expression correlates with the proliferative state of HL-60 cells, rather than with the differentiated state.     

Metabolism and activation of cyclopenta polycyclic aromatic hydrocarbons in isolated human lymphocytes, HL-60 cells and exposed rats

The metabolism of radiolabelled benz(j)aceanthrylene (B(j)A) was studied in suspensions of isolated human peripheral mononuclear blood cells (lymphocytes), using high performance liquid chromatography (HPLC). The only known metabolite found after 24 h exposure to 30 microg/ml (120 microM) B(j)A, was B(j)A-1,2-dihydrodiol, representing approximately 35% of the total metabolites formed. B(j)A, benz(l)aceanthrylene (B(l)A) and benzo(a)pyrene (B(a)P) all caused DNA adducts in human lymphocytes, as well as in the human promyelocytic cell line HL-60 cells, as measured by the 32P-postlabelling technique (30 microg/ml, 24 h). The total DNA adduct levels in human lymphocytes exposed to B(j)A, B(l)A or B(a)P were 0.13 +/- 0.03, 1.10 +/- 0.62 and 0.37 +/- 0.10 fmol/microg DNA, respectively, and similar levels were obtained in HL-60 cells (0.18 +/- 0.14, 0.97 +/- 0.35 and 0.29 +/- 0.17 fmol/microg DNA, respectively). For each compound, the human lymphocytes and HL-60 cells in addition showed similar DNA adduct patterns. Cells exposed to B(j)A revealed only one DNA adduct, which, judged by its TLC properties, resulted from B(j)A-1,2-epoxide. As measured by the alkaline filter elution technique, only low levels of single strand DNA breaks (SSB) were observed in both human lymphocytes and HL-60 cells after exposure to B(j)A, B(l)A or B(a)P (24 h, 30 microg/ml). By adding cytosine-1-beta-D-arabinofuranoside (Are C) and hydroxyurea (HU) 1 h prior to analysis to prevent strand break rejoining, some increase in SSB (2-3 times) was observed in the lymphocytes. Co-incubation of human lymphocytes with liver microsomes from PCB-treated rats for 1 h and exposure to B(j)A or B(l)A, increased the DNA adduct levels in the lymphocytes to 12.3 and 37.8 fmol/microg DNA, respectively. A large increase in SSB was also observed, whereas no such increase was observed after co-incubation with human liver microsomes. In vivo exposure of rats to 30 mg/kg B(j)A (i.p.) for 24 h revealed one major DNA adduct in lymphocytes and lung tissue (only one of three rats showed an adduct in liver tissue), apparently resulting from B(j)A-1,2-epoxide. The total DNA adduct level in the lymphocytes was 0.09 fmol/microg DNA, and in lung tissue between 0.10 and 0.62 fmol/microg DNA. Overall, the present data suggests that oxidation at the cyclopenta-ring is an important activation pathway for B(j)A in rats as well as in humans.     

Evidence for the presence of a kininogen-like species in a case of total deficiency of low and high molecular weight kininogens

EBI1-ligand chemokine (ELC) is a CC chemokine constitutively expressed in various lymphoid tissues and a high-affinity functional ligand for EBI1/CCR7, a seven transmembrane G-protein-coupled receptor originally identified as an Epstein-Barr virus (EBV)-inducible gene. Here we examined chemotactic activity of ELC on peripheral blood leukocytes. ELC attracted both CD4+ and CD8+ T cells, particularly efficiently after activation with IL-2 or with phytohemagglutinin (PHA) plus IL-2, as well as CD19+ B cells, but not CD16+ NK cells, CD14+ monocytes or neutrophils. Among CD3+ T cells, ELC attracted both CD45RO- naive and CD45RO+ memory subsets. ELC also induced vigorous calcium mobilization in T cells stimulated with IL-2 with an ED50 of 3 nM. ELC fused with the secreted form of alkaline phosphatase (ELC-SEAP) specifically bound to lymphocytes and this binding was blocked only by ELC among 10 CC chemokines so far tested. Notably, lymphocytes stimulated with IL-2 or T cells expanded by PHA plus IL-2 showed much higher levels of binding than fresh lymphocytes. Consistently, CCR7 mRNA was detected in CD4+ and CD8+ T cells as well as B cells, but not in NK cells, monocytes or neutrophils, and was dramatically increased in T cells upon treatment with IL-2 or with PHA plus IL-2. Like ELC mRNA, CCR7 mRNA was expressed in various lymphoid tissues. By in situ hybridization, ELC and CCR7 mRNA were detected in the parafollicular and inner cortical regions of a lymph node, and in the parafollicular regions of an appendix. Collectively, ELC and CCR7 may be involved in the trafficking of a broad spectrum of lymphocytes, especially activated T cells, into and within various lymphoid tissues.     

Intramembrane receptor-receptor interactions: integration of signal transduction pathways in the nervous system

Based on a hypothesis that post-mortem cellular (chiefly nuclear) changes in the white blood cells could reliably be correlated with the time interval since death, (ti), serial observations were made on the counts (total, differential) and light-microscopically observable 'degenerations' of white blood cells obtained from 30 non-refrigerated cadavers (experimental group) and similar cells obtained from 200 hospital patients (control group). While neutrophils degenerated rapidly, lymphocytes did so slowly; the eosinophils and monocytes degenerated at rates between these extremes. In cadaveric blood total counts of identifiable leucocytes on average dropped to zero by 84 hours, identifiable eosinophils and monocytes were first to 'disappear' (by 60 hours), followed by neutrophils (by 66 hours), and finally lymphocytes: identifiable lymphocytes disappeared completely at or around 84 hours from the time of death. This 'differential degeneration' was surprising but useful. Based on the use of all four characteristics--total and differential white cell counts, differential degeneration and morphology of cells--a method for a reasonably exact estimation of ti is presented. The method is appropriate for ti up to 84 hrs (3 1/2 days). Zero white cell counts (total, differential) and bizarre morphology (unidentifiable white blood cells) indicate a ti > 84 hrs. Avenues for further research are indicated.