Production of macrophage activating factor by human leukemic T cell lines

Human leukemic T cell lines were tested for their ability to produce a macrophage activating factor. When mouse peritoneal macrophages were cultured for 48 hr in the presence of culture supernatants from cell lines HPB-ALL, CCRF-CEM, or MOLT-4, glucose oxidation via the hexose monophosphate pathway was enhanced by five to seven fold. Culture supernatants from cell line HPB-MLT stimulated the oxidation to a lesser extent. However, cell line CCRF-HSB-2 was essentially inactive as a producer. The active supernatants also stimulated the release of hydrogen peroxide from macrophages, whereas the inactive one did not. Since treatment of the cell lines with 12-o-tetradecanoyl phorbol acetate or phytohemagglutinin had little effect on the production of the factor except HPB-ALL, the cell lines seemed to secrete the factor constitutively. The stimulatory effect was dose-dependent and evident at a concentration as low as a 1/80 dilution. The factor was resistant to heat treatment at 100 C for 20 min, nondialysable and sensitive to protease digestion. The activating factor could be partially purified by anion exchange and gel filtration chromatographies.     

Accessory function and interleukin 1 production by human leukemic cell lines

Highly purified T lymphocytes do not proliferate in response to mitogens, unless adherent HLA-DR-positive monocytes are added to the culture. This accessory function (AF) of monocytes requires the release of interleukin 1 (IL 1). Cells from three human leukemic cell lines, K562, HL60, and U937, could very efficiently replace monocytes in a 72-hr mitogen-induced T cell proliferation assay. The AF was clearly related to precise maturational stages of these cells; the hematopoietic precursor K562 cells spontaneously exerted high AF, but lost this property when treated with differentiation inducers. On the contrary, the promyelocytic HL60 cells and the "histiocytic" U937 cells exhibited no spontaneous AF, but acquired this property when induced to differentiate along the granulocytic and/or monocytic pathway. Three leukemic cells could not only stimulate T cells to proliferate and produce IL 2 in the presence of mitogens, but also under appropriate culture conditions these cells could produce IL 1, which could not be distinguished from normal human monocyte derived IL 1 by gel filtration and isoelectric focusing. Moreover, analysis of phenotypic markers revealed that AF and production of IL 1 could be demonstrated in different cell types and therefore are not restricted to the monocytic lineage. No HLA-DR antigen could be detected on K562 and HL60 cells. Thus, the expression of the DR antigens is not required for AF and IL 1 production in response to mitogens. These three human leukemic cell lines will provide convenient sources of human IL 1.     

Artificial ion channels formed by a synthetic cyclic peptide.

A new cyclic peptide 1 having an (LLLD)3 configuration pattern was designed that is capable of forming artificial transmembrane ion channels by self-assembly of planar peptide rings, with hydrophilic groups arrayed in the interior of the channel. Ion permeability in the presence of the synthetic peptide 1, cyclo[-Trp-Dap-Leu-D-Ala-Trp-Ser-Val-D-Ala-Trp-Ser-Ile-Gly-] (Dap: L-diaminopropionic acid), was observed in lipid bilayer membranes. The pH dependence of ionic conductance showed that the beta-amino group of Dap may play a role in the conductance of the peptide channels. Fourier-transform infrared and circular dichroism data imply that, in a membrane, a stack of cyclic peptides is formed in which the inter peptide H bonds form a kind of beta-structure analogous to that in the gramicidin A dimer and distinct from the H-bonding pattern of the beta-barrels.     

Sensitization to the hyperthermic and catecholamine-releasing effects of morphine

Rats were given alternating injections (one per day) of morphine (5 mg/kg) and saline, each substance paired with a distinct set of environmental cues. Over the course of nine injections of the drug, the hyperthermic response to morphine gradually increased. On the tenth such exposure to morphine, half of the rats were injected in the presence of cues previously paired with the drug, and half were injected in the presence of saline cues. In the former (drug-cue) group, hyperthermia, and plasma norepinephrine and epinephrine levels were significantly greater than in rats receiving morphine for the first time. The latter (saline-cue) group showed an intermediate thermic and catecholamine response, not statistically different from drug-cue or control animals. Under these conditions, the enhanced hyperthermic response to morphine (which has been variously described as sensitization or tolerance), was found to be accompanied by a similar increase in elevation of plasma catecholamines.     

The fibronectin-derived anti-adhesive peptide III14-2 suppresses adhesion and apoptosis of leukemic cell lines through down-regulation of protein-tyrosine phosphorylation.

We previously found that fibronectin (FN) has a cryptic functional site (YTIYVIAL, #1848-1855) opposing to cell adhesion to extracellular matrix (ECM). The present study demonstrates that the FN peptide containing this anti-adhesive site, termed peptide III14-2, affects programmed cell death (PCD) (apoptosis) as well as cell adhesion by down-regulating protein-tyrosine phosphorylation. Peptide III14-2 suppressed the integrin alpha5beta1-mediated adhesion of leukemic cell lines (K562 and HL60), and protein tyrosine phosphatase inhibitor, 1 microM phenylarsine oxide (PAO) blocked the anti-adhesive effect of peptide III14-2. These leukemic cells underwent PCD when exposed to PAO at the higher concentration (5 microM), as judged by nuclear and DNA fragmentations, and which was reversed by tyrosine kinase inhibitor, genistein. Peptide III14-2 suppressed the PAO-induced PCD, whereas a control peptide in which the anti-adhesive sequence YTIYVIAL is scrambled, was inactive. Western blotting showed that PAO stimulated the tyrosine phosphorylation of cellular proteins including focal adhesion kinase and that peptide III14-2 inhibited them, suggesting that protein-tyrosine phosphorylation represents a common early signal for the adhesion and PCD. The anti-adhesive site of FN molecule may play a crucial role also in a variety of cellular processes other than adhesion and PCD by down-regulating protein-tyrosine phosphorylation.     

The role of calcium in differentiation of leukemic cell lines

Increased calcium influx associated with differentiation of four human myeloid leukemic cell lines: HL-60, KG-1, U-937 and K-562, to either monocytic or granulocytic direction was demonstrated. Calcium influx was measured employing two methods; measurement of radioactive calcium influx rate at 4 degrees C and employing the fluorescent probe, fura-2 acetoxymethyl ester. The increase in Ca2+ influx was demonstrated with three chemically unrelated differentiation inducers: retinoic acid, 1 alpha, 25 dihydroxy vitamin D3 and dimethyl sulfoxide. Inhibitors of calcium uptake such as verapamil diltiazem and cromolyn, partially reduced differentiation, suggesting that differentiation of myeloid leukemic cell lines is dependent on the availability of extracellular calcium.     

Activation of human neutrophils by a synthetic anti-microbial peptide, KLKLLLLLKLK-NH2, via cell surface calreticulin.

We previously reported that a synthetic anti-bacterial peptide, KLKLLLLLKLK-NH2 (L5), showed significant chemotherapeutic activity in methicillin-resistant Staphylococcus aureus-infected mice, and its ability to activate human neutrophils was related to its chemotherapeutic activity. In this study, we found that activation of neutrophils by L5 was inhibited by pertussis toxin, suggesting that GTP-binding protein (G-protein) participates in this process. We isolated an L5-binding protein, which turned out to be human calreticulin, with a molecular mass of 60 kDa from neutrophil membranes. From experiments using an anti-calreticulin antibody, we proposed that calreticulin is partly localized on the surface of neutrophils, and L5-bound calreticulin transmits a signal into cells via G-protein to activate neutrophils to generate superoxide anion.     

In vivo induction of cytotoxic T cell response by a free synthetic peptide requires CD4+ T cell help.

Most attempts to induce CTL responses by in vivo priming with free synthetic peptides have been unsuccessful so far. However, two separate studies have recently succeeded in inducing antiviral CTL responses by immunizing mice with unmodified free synthetic peptides derived from nucleoproteins from either lymphocytic choriomeningitis virus or Sendai virus. In the present study, we have analyzed the cellular mechanisms by which the lymphocytic choriomeningitis virus synthetic peptide induced CTL responses. We demonstrated that this peptide, which was previously shown to be recognized by CD8+ T cells, also contains a helper CD4+ T cell epitope. It stimulates in vivo both CD4+ T cell-mediated CTL response. The in vivo elimination of CD4+ T cells by treatment with a mAb was shown to strongly reduce the antipeptide CTL response. This study therefore demonstrates that to be able to induce CTL responses, a peptide has to stimulate both CD4+ and CD8+ T cell subset.     

Differentiation-associated changes of cation-transport activities in myeloid leukemic cell lines

Induction of differentiation in HL-60 and U-937 leukemic cell lines, resulted in 1.5-10-fold increase in 45Ca2+ uptake. The increased 45Ca2+ uptake in the differentiating cells was inhibited by verapamil, cromolyn and amiloride. Elevation in Ca2+ uptake in differentiating cells was also demonstrated using the fluorescent probe, fura-2 acetoxymethyl ester. The increased 45Ca2+ uptake was accompanied by a decrease in ouabain-insensitive and -sensitive 86Rb+ uptake. Furthermore, correlation between the changes in these activities was observed. Modulation of extracellular pH affected differentiation: higher pH increased the extent of differentiation.     

Induction of hepatitis A virus-neutralizing antibody by a virus-specific synthetic peptide.

Comparative surface feature analyses of the VP1 sequences of hepatitis A virus (HAV) and poliovirus type 1 allowed an alignment of the two sequences and an identification of probable HAV neutralization antigenic sites. A synthetic peptide containing the HAV-specific amino acid sequence of one of these sites induced anti-HAV-neutralizing antibodies. It is concluded that a structural homology exists between the two viruses, despite minimal primary sequence conservation.     

Phosphorylation of a synthetic gastrin peptide by the tyrosine kinase of A431 cell membranes

The peptide Arg.Arg.Leu.Glu.Glu.Glu.Glu.Glu.Ala.Tyr.Gly was synthesized as an analogue of residues 20–30 of human gastrin 34. The epidermal growth factor-stimulated tyrosine kinase of A431 cell membranes phosphorylated the peptide's single tyrosine residue. K_m values of 0.11 and 0.61mM and V_max values of 1.71 and 0.68nmol/min/mg were obtained in the presence and absence of epidermal growth factor respectively. This is the first report of phosphorylation of tyrosine in a sequence related to a human hormone.     

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.     

Inhibition of epidermal growth factor/transforming growth factor-alpha-stimulated cell growth by a synthetic peptide

Estrogen-stimulated growth of the human mammary adenocarcinoma cell line MCF-7 is significantly inhibited by monoclonal antibodies to the epidermal growth factor (EGF) receptor that act as antagonists of EGF's mitogenic events by competing for high-affinity EGF receptor binding sites. These antibodies likewise inhibit the EGF or transforming growth factor-alpha (TGF-alpha)-stimulated growth of these MCF-7 cells. An analogous pattern of specific EGF or TGF-alpha growth inhibitory activity was obtained using a synthetic peptide analog encompassing the third disulfide loop region of TGF-alpha, but containing additional modifications designed for increased membrane affinity [( Ac-D-hArg(Et)2(31),Gly32,33]HuTGF-alpha(31-43)NH2). The growth factor antagonism by this synthetic peptide was specific in that it inhibited EGF, TGF-alpha, or estrogen-stimulated growth of MCF-7 cells but did not inhibit insulin-like growth factor-1 (IGF-1)-stimulated cell growth. Altogether, these results suggest that a significant portion of the estrogen-stimulated growth of these MCF-7 cells is mediated in an autocrine/paracrine manner by release of EGF or TGF-alpha-like growth factors. The TGF-alpha peptide likewise inhibited EGF- but not fibroblast growth factor (FGF)- or platelet-derived growth factor (PDGF)-stimulated growth of NIH-3T3 cells in completely defined media; but had no effect on growth or DNA synthesis of G0-arrested cells, nor did it effect growth of NR-6 cells, which are nonresponsive to EGF. Although this synthetic peptide did not directly compete with EGF for cell surface receptor binding, it exhibited binding to a cell surface component (followed by internalization), which likewise was not competed by EGF. The peptide did not directly inhibit EGF-stimulated phosphorylation of the EGF receptor, nor did it inhibit phosphorylation of an exogenous substrate, angiotensin II, by activated EGF receptor. The TGF-alpha peptide did, however, affect the structure of laminin as manifested by laminin self-aggregation; this affect on laminin may, in turn, have a modulatory effect on EGF-mediated cell growth.     

Synthesis of Bcl-2 in response to anthracycline treatment may contribute to an apoptosis-resistant phenotype in leukemic cell lines.

BACKGROUND: Some forms of chemoresistance in leukemia may start from failure of tumour cells to successfully undergo apoptosis and Bcl-2 may play a role in this defect. Therefore, we evaluated the Bcl-2 content and synthesis in relation with the apoptotic potential in leukemic cell lines after anthracycline treatment. METHODS: U937, HL60, and K562 cells and their drug resistant (DR) variants were treated with varying concentrations of Idarubicin (IDA). Apoptosis was evaluated by fluorescence microscopy after acridine orange staining. Bcl-2 and Bax content were evaluated either by flow cytometry after indirect immunolabelling or by Western blot. RESULTS: High Bcl-2 contents were not related to a poor ability to undergo apoptosis in U937, HL60, K562 and their DR variants. IDA induced a concentration-dependent increase in Bcl-2 content in all cell lines as long as they do not perform apoptosis. Enhanced Bcl-2 expression was inhibited by cycloheximide, actinomycin D, or antisense oligonucleotide directed against bcl-2 mRNA. Bcl-2 expression was also increased in the resistant U937 variant after serum deprivation or C2-ceramide treatment. The synthesis of Bcl-2 led to an increased Bcl-2/Bax ratio solely in the cells with an apoptosis-resistance phenotype. CONCLUSIONS: These data suggest that exposure to IDA induces Bcl-2 expression in leukemic cell lines, and that this mechanism could contribute to apoptosis resistance and participate in the acquisition of chemoresistance. They also confirm that the evolution of the Bcl-2/Bax ratio reflects apoptotic ability better than the steady state level of Bcl-2 expression.     

Mithramycin A activates Fas death pathway in leukemic cell lines

Mithramycin A (MMA, trade name Plicamycin) can facilitate TNFα- (Tumor Necrosis Factor) and Fas ligand-induced apoptosis. Besides, several drugs play their anticancer effect through Fas apoptotic pathway. So we investigated the effect of MMA on Fas signaling. In this study we show that MMA induces apoptosis in Fas sensitive Jurkat cells and Fas resistant KG1a cells. This effect involves Fas apoptotic pathway: cell exposure to MMA leads to Fas clustering at the cell surface, DISC (Death Inducing Signaling Complex) formation and caspase cleavage. This phenomenon is independent of Fas ligand/Fas interaction and blockade of Fas death pathway partially inhibits MMA-induced apoptosis. Moreover the activation of Fas apoptotic pathway by MMA is correlated to the modulation of c-Flip_L expression. Finally, pre-treatment with sub-lethal doses of MMA sensitizes KG1a cells to chemotherapeutic agents. Thus all these results may have important implications to improve clinical treatments.     

pH-dependent fusion of phosphatidylcholine small vesicles. Induction by a synthetic amphipathic peptide

A synthetic, amphipathic 30-amino acid peptide with the major repeat unit Glu-Ala-Leu-Ala (GALA) was designed to mimic the behavior of the fusogenic sequences of viral fusion proteins. GALA is a water-soluble peptide with an aperiodic conformation at neutral pH and becomes an amphipathic alpha-helix as the pH is lowered to 5.0 where it interacts with bilayers. Fluorescence energy transfer measurements indicated that GALA induced lipid mixing between phosphatidylcholine small unilamellar vesicles but not large unilamellar vesicles. This lipid mixing occurred only at pH 5.0 and not at neutral pH. Concomitant with lipid mixing, the vesicles increased in diameter from 500 to 750 to 1000 A as measured by dynamic light scattering and internal volume determination. GALA induced leakage of small molecules (Mr 450) at pH 5.0 was too rapid to permit detection of contents mixing. However, retention of larger molecules (Mr 4100) under the same conditions suggests that vesicle fusion is occurring. For a 100/1 lipid/peptide ratio all vesicles fused just once, whereas for a 50/1 ratio higher order fusion products formed. A mass action model gives good simulation of the kinetics of increase in fluorescence intensity and yields rate constants of aggregation and fusion. As the lipid to peptide ratio decreases from 100/1 to 50/1 both rate constants of aggregation and fusion increase, indicating that GALA is a genuine inducer of vesicle fusion. The presence of divalent cations which can alter GALAs conformation at pH 7.5 had little effect on its lipid mixing activity. GALA was modified by altering the sequence while keeping the amino acid composition constant or by shortening the sequence. These peptides did not have any lipid mixing activity nor did they induce an increase in vesicle size. Together, these results indicate that fusion of phosphatidylcholine small unilamellar vesicles induced by GALA requires both a peptide length greater than 16 amino acids as well as a defined topology of the hydrophobic residues.     

Loss and reappearance of transferrin receptors in human leukemic cell lines

Serum transferrin (the iron binding protein) exerts its iron carrier function at the cell surface after binding to the appropriate receptor (TrR). In this work it is demonstrated that differentiating agents induce loss of TrR from the surface of three leukemic cell lines (Molt-3, HL-60 and K-562). Loss of TrR correlates with change in morphology and induction of phenotypic markers of the differentiated cells. Removal of the differentiating agent from the culture is followed by reexpression of TrR on the cell surface. The data presented in this paper suggest that TrR may play a regulatory role in cell differentiation and malignant transformation.     

Mycoplasma contamination of leukemic cell lines alters protein expression determined by reverse phase protein arrays

Mycoplasma contamination is a major problem in cell culturing, potentially altering the results of cell line-based experiments in largely uncharacterized ways. To define the consequences of mycoplasma infection at the level of protein expression we utilized the reverse phase protein array technology to analyze the expression of 235 proteins in mycoplasma infected, uninfected post treatment, and never-infected leukemic cell lines. Overall, protein profiles of cultured cells remained relatively stable after mycoplasma infection. However, paired comparisons for individual proteins identified that 18.7% of the proteins significantly changed between the infected and the never-infected cell line samples, and that 14.0% of the proteins significantly altered between the infected and the post treatment samples. Six percent of the proteins were affected in the post treatment samples compared to the never-infected samples, and 7.2% compared to treated cells that had never had mycoplasma infection before. Proteins that were significantly altered in the infected cells were enriched for apoptotic signaling processes and auto-phosphorylation, suggesting an increased cellular stress and a decreased growth rate. In conclusion, this study shows that mycoplasma infection of leukemic cell lines alters the proteins expression levels, potentially confounding experimental results. This reinforces the need for regular testing of mycoplasma.     

Apoptotic events induced by synthetic naphthylchalcones in human acute leukemia cell lines

Acute leukemia is a disorder of the hematopoietic system characterized by the expansion of a clonal population of cells blocked from differentiating into mature cells. Recent studies have shown that chalcones and their derivatives induce apoptosis in different cell lines. Since new compounds with biological activity are needed, the aim of this study was to evaluate the cytotoxic effect of three synthetic chalcones, derived from 1-naphthaldehyde and 2-naphthaldehyde, on human acute myeloid leukemia K562 cells and on human acute lymphoblastic leukemia Jurkat cells. Based on the results, the most cytotoxic compound (A1) was chosen for further analysis in six human acute leukemia cells and in a human colon adenocarcinoma cell line (HT-29). Chalcone A1 significantly reduced the cell viability of K562, Jurkat, Kasumi, U937, CEM and NB4 cells in a concentration and time-dependent manner when compared with the control group (IC₅₀ values between ∼1.5 μM and 40 μM). It was also cytotoxic to HL-29 cells. To further examine its effect on normal cells, peripheral blood lymphocytes collected from healthy volunteers were incubated with the compound. It has also been incubated with human fibroblasts cultured from bone marrow (JMA). Chalcone A1 is non-cytotoxic to PBL cells and to JMA cells. A1 caused significant cell cycle arrest in all phases according to the cell line, and increased the proportion of cells in the sub G0/G1 phase. To evaluate whether this chalcone induced cell death via an apoptotic or necrotic pathway, cell morphology was examined using fluorescence microscopy. Cells treated with A1 at IC₅₀ demonstrated the morphological characteristic of apoptosis, such as chromatin condensation and formation of apoptotic bodies. Apoptosis was confirmed by externalization of phosphatidylserine, which was detected by the Annexin V-FITC method, and by DNA fragmentation. The results suggest that chalcone A1 has potential as a new lead compound for cancer therapy.