Membrane action of DMSO and other chemical inducers of Friend leukaemic cell differentiation

DMSO and other cryoprotective agents produce a pronounced increase on the phase transition temperature of phospholipid membranes, indicating an increased stability. The effects of DMSO and other cryoprotective agents, divalent cations, and local anaesthetics on the transition temperature of phospholipid membranes seem to correlate with their effects on the differentiation of Friend leukaemic cells in vitro. These studies suggest that the induction of differentiation by cryoprotective agents may be the result of the interaction of these agents with cell membranes.     

Phospholipid composition of the mammalian red cell membrane can be rationalized by a superlattice model

Although the phospholipid composition of the erythrocyte membrane has been studied extensively, it remains an enigma as to how the observed composition arises and is maintained. We show here that the phospholipid composition of the human erythrocyte membrane as a whole, as well as the composition of its individual leaflets, is closely predicted by a model proposing that phospholipid head groups tend to adopt regular, superlattice-like lateral distributions. The phospholipid composition of the erythrocyte membrane from most other mammalian species, as well as of the platelet plasma membrane, also agrees closely with the predictions of the superlattice model. Statistical analyses indicate that the agreement between the observed and predicted compositions is highly significant, thus suggesting that head group superlattices may indeed play a central role in the maintenance of the phospholipid composition of the erythrocyte membrane.     

Regulation of T helper cell differentiation in vivo by soluble and membrane proteins provided by antigen-presenting cells

The aim of this study was to test whether the nature of the antigen-presenting cell (APC) can influence the Th1/Th2 balance in vivo. Our data show that dendritic cells (DC), pulsed extracorporeally with antigen, induced the development of cells secreting IL-2, IFN-gamma and IL-4 upon antigen rechallenge in vitro. Priming with peritoneal macrophages sensitized cells that produced IL-4 but not IFN-gamma. To identify the factors involved in T helper development, mice were primed with APC with or without treatment with neutralizing antibodies to costimulatory molecules or cytokines. Our results indicate that priming with DC or macrophages is strictly dependent on the CD28-CTLA4/B7 interaction. Of note, CD86 provides the initial signal to induce naive T cells to become IL-4 producers, whereas CD80 is a more neutral differentiation signal. IL-12, released by the DC, appears as a potent and obligatory inducer of differentiation for IFN-gamma-producing cells. IL-6, although produced by both APC populations, is necessary to direct activation of the Th2-type response by macrophages but not by DC.     

Drosophila syntaxin is required for cell viability and may function in membrane formation and stabilization

The role of the Drosophila homologue of syntaxin-1A (syx) in neurotransmission has been extensively studied. However, developmental Northern analyses and in situ hybridization experiments show that SYX mRNA is expressed during all stages and in many tissues. We have isolated new mutations in syx that reveal roles for syx outside the nervous system. In the ovary, SYX is present in the germarium, but it is predominantly localized to nurse cell membranes. Mitotic recombination experiments in the germline show SYX is essential for oogenesis and may participate in membrane biogenesis in the nurse cells. In the early embryo, a large contribution of maternally deposited RNA is present, and the protein is localized at cell membranes during cellularization. After the maternal contribution is depleted, zygotically produced SYX assists secretion events occurring late in embryogenesis, such as cuticle deposition and neurotransmitter release. However, SYX is also required in larval imaginal discs, as certain hypomorphic mutant combinations exhibit rough eyes and wing notch defects indicative of cell death. Furthermore, recombinant clones that lack syx cause cell lethality in the developing eye. We propose that, similar to its roles in cuticle secretion and neurotransmitter release, SYX may mediate membrane assembly events throughout Drosophila development.     

Autoinduction of differentiation in myeloid leukemic cells: restoration of normal coupling between growth and differentiation in leukemic cells that constitutively produce their own growth-inducing protein

Growth and differentiation of normal myeloid haematopoietic cells are regulated by a family of macrophage- and granulocyte-inducing (MGI) proteins. Some of these proteins (MGI-1) induce cell growth and others (MGI-2) induce cell differentiation. Addition of MGI-1 to normal myeloid cells induces growth and also induces the endogenous production of MGI-2. This induction of differentiation-inducing protein by growth-inducing protein then ensures the coupling between growth and differentiation found in normal cells. There are myeloid leukemic cells that constitutively produce their own MGI-1, but the cells do not differentiate in culture medium containing horse or calf serum. By removing serum from the medium, or in medium with mouse or rat serum, these leukemic cells are induced to differentiate to mature cells, which like normal mature cells, then no longer multiply. Leukemic cells with constitutive production of MGI-1 continuously cultured in serum-free medium with transferrin were also induced to differentiate by removing transferrin. This induction of differentiation was in all these cases associated with the endogenous production of MGI-2 by the cells. The results indicate that changes in specific constituents of the culture medium can result in autoinduction of differentiation in these leukemic cells due to restoration of the induction of MGI-2 by MGI-1, which then restores the normal coupling of growth and differentiation.     

Association of host cell surface adhesion receptors and other membrane proteins with HIV and SIV

We have developed a MAb-based capture assay to study the association of host cell membrane proteins with HIV and SIV. Class I and II MHC proteins were found to be associated with HIV as previously described. In addition to these molecules a number of other host molecules were found to be acquired by HIV, including CD71, CD63, CD43, and CD8. We have demonstrated that the major leukocyte adhesion receptors LFA-1 (CD11A/CD18) and CD44 are also associated with HIV. The level of surface expression of host membrane proteins did not predict relative expression (capture efficiency) of the virus. The use of virus-susceptible indicator cells showed that the assay involved host membrane protein-mediated capture of infectious HIV and SIV particles. Our data indicate that HIV and SIV acquire a number of host membrane proteins including adhesion receptors and that this process may be nonrandom. The acquisition of host cell adhesion receptors by HIV and SIV could have profound effects on the biology of the viruses, including binding, infectivity, and tropism.     

Phospholipid dynamics and function in cell membrane of blood cells. With reference to mode of action of certain amphiphilic drugs

An asymmetric modification of the cell membrane lipid bilayer of erythrocytes by means of exogenous phospholipase (PL) treatment or of phospholipid addition induced an echinocyte- or stomatocyte-type shape change of the cells. Such membrane shape change was demonstrated to be due to asymmetric swelling or shrinkage of the lipid bilayer halves, from the simultaneous determination of the change or intra-bilayer transfer of the phospholipid molecules. In the collagen-induced activation of blood platelets, the importance of PLA2 activation as the initial reaction of the signaling system was demonstrated, and the regulatory effects of the membrane fluidity change on the enzyme activation were also shown. In the antigen-induced activation of peritoneal mast cells, a pathway through the PLD activation appeared to be more important. Phosphatidic acid produced from the membrane phospholipids through the PLD activation was shown to act as an intracellular mediator to stimulate the PLA2 activation. Various amphiphilic drugs, including benzylisoquinoline alkaloids such as cepharanthine, were shown to inhibit the platelet activation and the mechanism was studied. The anti-platelet activity appeared to be mainly due to inhibition of the PLA2 activation.     

Role of mitochondrial membrane potential in the regulation of murine erythroleukemia cell differentiation

The level of cytoplasmic calcium ions appears to be important in the control of murine erythroleukemia (MEL) cell differentiation. Our interest in this study focuses on the relationship between the regulation of calcium concentration and differentiation. We used the fluorescent membrane probe DiOC6 to examine the relationship between MEL cell mitochondria and changes in cytoplasmic calcium levels occurring at the initiation of commitment. Fluorescence microscopy reveals the selective association of DiOC6 with MEL cell mitochondria, where an enhanced fluorescence is observed. Treatment of cells with dimethylsulfoxide (DMSO) or other inducers causes a decrease in mitochondria-associated fluorescence levels that occurs with the initiation of commitment. A decrease in DiOC6 fluorescence is caused by agents that reduce mitochondrial membrane potential, but is only slightly affected by agents that alter plasma membrane potential. Amiloride and EGTA, agents that prevent commitment and inhibit calcium uptake, also prevent the decrease in DiOC6 uptake caused by DMSO. The effect of DMSO on MEL cell mitochondria is mimicked by FCCP, a proton ionophore that dissipates mitochondrial membrane potential. FCCP also caused MEL cell mitochondria to release calcium into the cytoplasm. When MEL cells are treated with DMSO plus FCCP, commitment is initiated without the lag period observed when cells are treated with DMSO alone. These results are consistent with the hypothesis that mitochondrial transmembrane potential is important in the regulation of cytoplasmic calcium levels at the time of commitment of MEL cells to terminal differentiation.     

19-nor vitamin-D analogs: a new class of potent inhibitors of proliferation and inducers of differentiation of human myeloid leukemia cell lines

We have studied the in vitro biological activities and mechanisms of action of 1,25-dihydroxyvitamin D3 (1,25D3) and nine potent 1,25D3 analogs on proliferation and differentiation of myeloid leukemia cell lines (HL-60, retinoic acid-resistant HL-60 [RA-res HL-60], NB4 and Kasumi-1). The common novel structural motiff for almost all the analogs included removal of C-19 (19-nor); each also had unsaturation of the side chain. All the compounds were potent; for example, the concentration of analogs producing a 50% clonal inhibition (ED50) ranged between 1 x 10(-9) to 4 x 10(-11) mol/L when using the HL-60 cell line. The most active compound [1, 25(OH)2-16,23E-diene-26-trifluoro-19-nor-cholecalciferol (Ro 25-9716)] had an ED50 of 4 x 10(-11) mol/L; in contrast, the 1,25D3 produced an ED50 of 10(-9) mol/L with the HL-60 target cells. Ro 25-9716 (10(-9) mol/L, 3 days) was a strong inducer of myeloid differentiation because it caused 92% of the HL-60 cells to express CD11b and 75% of these cells to reduce nitroblue tetrazolium (NBT). This compound (10(-8) mol/L, 4 days) also caused HL-60 cells to arrest in the G1 phase of the cell cycle (88% cells in G1 v 48% of the untreated control cells). The p27(kip-1), a cyclin-dependent kinase inhibitor which is important in blocking the cell cycle, was induced more quickly and potently by Ro 25-9716 (10(-7) mol/L, 0 to 5 days) than by 1,25D3, suggesting a possible mechanism by which these analogs inhibit proliferation of leukemic growth. The NB4 promyelocytic leukemia cells cultured with the Ro 25-9716 were also inhibited in their clonal proliferation (ED50, 5 x 10(-11) mol/L) and their expression of CD11b was enhanced (80% positive [10(-9) mol/L, 4 days] v 27% untreated NB4 cells). Moreover, the combination of Ro 25-9716 (10(-9) mol/L) and all-trans retinoic acid (ATRA, 10(-7) mol/L) induced 92% of the NB4 cells to reduce NBT, whereas only 26% of the cells became NBT positive after a similar exposure to the combination of 1,25D3 and ATRA. Surprisingly, Ro 25-9716 also inhibited the clonal growth of poorly differentiated leukemia cell lines (RA-res HL-60 [ED50, 4 x 10(-9) mol/L] and Kasumi-1 [ED50, 5 x 10(-10) mol/L]). For HL-60 cells, Ro 25-9716 markedly decreased the percent of the cells in S phase of the cell cycle and increased the expression of the cyclin-dependent kinase inhibitor, p27(kip-1). In summary, 19-nor vitamin D3 compounds strongly induced differentiation and inhibited clonal proliferation of various myeloid leukemia cell lines, suggesting a therapeutic niche for their use in myeloid leukemia.     

The effect of differentiation inducers on the integrin expression of K562 erythroleukemia cells

The DNA sequences of the recA gene from 25 strains of bacteria are known. The evolution of these recA gene sequences, and of the derived RecA protein sequences, is examined, with special reference to the effect of variations in genomic G + C content. From the aligned RecA protein sequences, phylogenetic trees have been drawn using both distance matrix and maximum parsimony methods. There is a broad concordance between these trees and those derived from other data (largely 16S ribosomal RNA sequences). There is a fair degree of certainty in the relationships among the "Purple" or Proteobacteria, but the branching pattern between higher taxa within the eubacteria cannot be reliably resolved with these data.     

Hemoglobin synthesis in cell hybrids formed between teratocarcinoma and Friend erythroleukemia cells

Viable hybrid cells have been isolated following fusion of Friend erythroleukemia cells and undifferentiated teratocarcinoma cells. The hybrids formed between near-diploid parental cells resembled Friend cells in the ability to grow in suspension and to synthesize hemoglobin in the presence of the chemical inducers dimethyl sulfoxide (DMSO) and ouabain. Erythropoietin (EPO) was effective in inducing hemoglobin synthesis in some of the hybrid cell lines. The hemoglobins synthesized by the hybrids were of the adult forms, but were quantitatively different from those hemoglobins synthesized by the parental Friend cells, suggesting that the fusion event modulated the expression of the hemoglobin chain genes.     

Resistance to apoptosis induced by serum depletion and all-trans retinoic acid in drug-resistant leukemic cell lines

The relation between resistance to anticancer drugs and resistance to apoptosis has been investigated in the human leukemic cell line(KY-821) and its drug-resistant sublines. Under serum depletion conditions, drug-resistant cell lines showed apoptotic resistance when compared with the parental cell line. Drug resistant cell lines also showed resistance to apoptosis when treated with all-trans retinoic acid. DNA fragmentation was low in drug resistant cell lines under both stimulations. Flowcytometry analysis did not show any alterations of the Fas antigen, p53, bcl-2 and c-myc protein expression toward inhibition of apoptotic response in drug-resistant sublines. These results indicate that drug-resistant leukemic cells still show resistance to apoptosis-inducing stimulation such as poor nutrition and differentiation-inducing agents.     

Alteration of cell membrane by stress waves in vitro

Experiments on the biological effects of laser-induced stress waves indicate that there is a transient increase in the permeability of the cell membrane. A cell viability assay (propidium iodide exclusion) shows that mouse breast sarcoma cells are viable after a stress wave. The kinetics of this transient membrane permeability are measured using time-resolved fluorescence imaging. The efflux of a membrane-impermeable fluorescent probe (calcein) following the application of a 300-bar stress wave implies that there is an increase in the membrane permeability. This efflux ceases within 80 s after a stress wave, suggesting that the membrane is no longer permeable to the fluorescent probe. Fitting the observed kinetics to a simple diffusion model yields an average initial diffusion constant of 2.2 +/- 1.3 x 10(-7) cm2/s for mouse breast sarcoma cells following the application of a laser-induced stress wave.