Induction by synthetic polyribonucleotide poly(I) of differentiation of cultured mouse myeloid leukemic cells.

The effects of some synthetic polyribonucleotides on induction of differentiation of mouse myeloid leukemic M1 cells were examined. Poly(I) was found to be a potent inducer; on treatment with 100--200 microgram/ml of poly(I) for 2--4 days, M1 cells differentiated into cells resembling macrophages and granulocytes and developed phagocytosis and locomotive activities, Fc receptors and lysozyme activity. Poly(C) was less effective than poly(I) for induction of phagocytic activity, while the other single-stranded RNAs, poly(U) and poly(A), had no effect. Double-stranded RNAs, such as poly(I) . poly(C) and poly(A) . poly(U), were cytotoxic to M1 cells, and differentiation of the cells could not be detected even at the highest tolerable concentrations of these double-stranded RNAs.     

Differential susceptibilities of DNA polymerases-alpha and -beta to polyanions.

The effects of various polyanions including synthetic polynucleotides on DNApolymerases-alpha and -beta from blastulae of the sea urchin Hemicentrotus pulcherrimus and HeLa cells were studied. Only DNA polymerase-alpha was inhibited by polyanions, such as polyvinyl sufate, dextran sulfate, heparin, poly(G), poly(I), poly(U) and poly(ADP-Rib). Of the various polynucleotides tested, poly(G) and poly(I) were the strongest inhibitors. Kinetic studies showed that the Ki value for poly(G) was 0.3 microgram/ml and that poly(G) had 20-fold higher affinity than activated DNA for the template-primer site of DNA polymerase-alpha. Poly(U) and poly(ADP-Rib) were also inhibitory, but they were one hundredth as inhibitory as poly(G) or poly(I). Poly(A), poly(C), poly(A).poly(U) AND POLY(I).poly(C) were not inhibitory to DNA polymerase-alpha. In contrast, DNA olymerase-beta was not affected at all by these polyanions under the same conditions.     

Interferon α selectively affects expression of the human myeloid cell nuclear differentiation antigen in late stage cells in the monocytic but not the granulocytic lineage

The human myeloid cell nuclear differentiation antigen (MNDA) is expressed constitutively in cells of the myeloid lineage, appearing in myeloblast cells in some cases of acute myeloid leukemia and consistently being detected in promyelocyte stage cells as well as in all later stage cells including peripheral blood monocytes and granulocytes. The human myeloid leukemia cell lines, HL-60, U937, and THP-1, express similar levels of immunochemically detectable MNDA. Although, the level of MNDA mRNA in primary monocytes is very low it was up-regulated at 6 h following the addition of interferon α. The effect of interferon α on the MNDA mRNA is also observed in the cell lines HL-60, U937, and THP-1. The MNDA mRNA level in primary granulocytes was unaffected by addition of interferon α and other agents including interferon γ, endotoxin, poly (I) · poly (C), and FMLP. The MNDA mRNA level in the myeloid cell lines was also unaffected by the latter four agents. Induction of differentiation in the myeloid cell lines with phorbol ester induces monocyte differentiation which was accompanied by a decrease in MNDA mRNA level. This reduced level of mRNA could then be elevated with subsequent interferon α treatment. The effects of phorbol ester on MNDA mRNA appeared to be associated with induced differentiation since inhibiting cell proliferation did not alter the level of MNDA mRNA and cell cycle variation in MNDA mRNA levels were not observed. The ability of interferon α to up-regulate MNDA mRNA in phorbol ester treated myeloid cell lines is consistent with the observations made in primary monocytes. Granulocyte differentiation induced by retinoic acid treatment of HL-60 cells did not alter the MNDA mRNA level which was also unchanged following subsequent treatment with interferon α. The lack of interferon α effects on retinoic acid treated HL-60 cells is consistent with its inability to influence MNDA mRNA level in primary granulocytes.     

Ganglioside GM3 as a modulator of differentiation of mouse myeloid leukemia cells (M1-T22)

The effects of exogenously added glycosphingolipids on the differentiation of mouse myeloid leukemia cells (M1-T22) have been studied. Eight gangliosides and ten neutral glycosphingolipids were tested in terms of their induction of phagocytic activities on the leukemia cells. N-Acetyl-neuraminosyllactosylceramide (NAc-GM3) was the most effective glycolipid for inducing the activity. By the addition of 25 micrograms/ml of NAc-GM3, about 70 percent of the cells acquired phagocytic activity within 20 h incubation. GM1a showed about half the activity of the GM3. In the case of the neutral glycosphingolipids, lactosylceramide (CDH) and globotriaosylceramide (CTH) showed significant effects on the induction of phagocytic activity. Preincubation of the cells with the NAc-GM3 enhanced the effect of dexamethasone as a differentiation inducer on M1-T22 cells. When a human promyelocytic leukemia cell line, HL-60, was preincubated with the NAc-GM3 ganglioside, induction of the phagocytic activity, together with inhibition of the cell growth by phorbol ester (TPA), were markedly enhanced. From these observations, the NAc-GM3 ganglioside seems to act as a modulator of differentiation of mouse myeloid leukemia cells and also of HL-60 cells.     

Enhancement by double-stranded polyribonucleotides of production by cultured mouse peritoneal macrophages of differentiation-stimulating factor(s) for mouse myeloid leukaemic cells.

Mouse peritoneal macrophages release a factor(s) that stimulates differentiation of a mouse myeloid leukaemic cell line into mature granulocytes and macrophages. Treatment of the macrophages with the synthetic double-stranded polyribonucleotides poly(I).poly(C) and poly(A).poly(U) resulted in enhanced release of the factor into the culture medium. The effect was maximal after treatment with polyribonucleotides for 1 h, and the optimal dose of poly(I).poly(C) was 50 microgram/ml. The single-stranded polyribonucleotides poly(I) and poly(C) at the same concentration were far less effective. The differentiation-stimulating factor was detected not only in the cultured medium but also in the cell lysate. Exposure of macrophages to poly(I).poly(C) enhanced the total activity of the factor in both the culture medium and the cell lysate. The effect of this compound was blocked by the presence of cycloheximide. These results suggest that double-stranded polyribonucleotides enhance production of the differentiation-stimulating factor by peritoneal macrophages.     

DNA topoisomerase I from calf thymus is inhibited in vitro by poly(ADP-ribosylation)

A slight DNA topoisomerase I activity was detected in highly purified poly(ADP-Rib)polymerase prepared from calf thymus. This copurified activity was found to be suppressed under conditions where the poly(ADP-ribosylation) reaction occurs in the presence of NAD. Purified topoisomerase I from calf thymus was shown to be ADP-ribosylated by poly(ADP-Rib) polymerase purified from the same tissue. Poly(ADP-ribosylation) of topoisomerase I produces an inhibition of the enzymatic activity in parallel to the extent of ADP-ribosylation. The fact that a slight poly(ADP-Rib) polymerase activity was also found to copurify with a topoisomerase I preparation and that topoisomerase I activity can be modified by ADP-ribosylation, may suggest a spatial and functional correlation of these two enzymes in chromatin.     

Endonucleolytic cleavage of murine leukemia virus 35S RNA by microsome-associated nuclease.

Moloney murine leukemia virus 35S RNA (molecular weight 3 to 3.4 × 10⁶) is cleaved by nuclease activity present in microsomal fractions from MLV infected or uninfected mouse embryo cells to two RNA species of approximate molecular weights 1.8 × 10⁶ and 1.5 × 10⁶. Microsomal fractions from MLV infected and uninfected cells also contained nucleolytic activity that solubilized [³H]poly(A)·poly(U) but not [³H]poly(C) or [³H]poly(U); the cleavage of poly(A)·poly(U) was inhibited by ethidium bromide. The cleavage of MLV RNA was also inhibited by ethidium bromide, suggesting double stranded regions in 35S RNA as the site of cleavage.     

Induction of differentiation of human and mouse myeloid leukemia cells by camptothecin

Low concentrations of camptothecin induced differentiation of human and mouse myeloid leukemia cells including human HL60, U937, ML1, and K562 cells and mouse M1 cells as measured by various differentiation-associated properties. When K562 cells were pretreated with 20 nM camptothecin for 2 h, 53% of the cells were induced to differentiate as measured by NBT staining. Significant single strand breaks in DNA of K562 cells were caused by this treatment. Most single strand breaks were accompanied by protein-DNA cross linking. The combination of camptothecin and rTNF synergistically induced differentiation of human ML1, U937, and M1 cells. These results suggest that topo I may be important in some differentiation of myeloid leukemia cells.     

Nuclear protein modification and chromatin substructure. 3. Relationship between poly(adenosine diphosphate) ribosylation and different functional forms of chromatin.

The relationship between poly(adenosine diphosphate) ribosylation of nuclear proteins and functionally different forms of chromatin from mid-S-phase HeLa nuclei was investigated. The major observations emerging from this study were that unique nonhistone proteins were modified in mid-S-phase HeLa nuclei. The major acceptor for poly(adenosine diphosphate-ribose) [poly(ADP-Rib)] was an internucleosomal nonhistone protein (protein C; 125 000 molecular weight). Histones H3, H1, H2b, and H2a but not H4 were ADP-ribosylated in S-phase nuclei. Chromatin fragments preferentially released by micrococcal nuclease were enriched in nonhistone proteins, poly(ADP)-ribosylated nuclear proteins, poly(ADP-Rib) polymerase activity and nascent DNA from the DNA replicating fork. In extended forms of chromatin, contiguous to the DNA replicating fork, poly(ADP-Rib) polymerase was maximally active. However, in chromatin distal to the replicating fork (i.e., more condensed structures), nucleosomal histones and histone H1 were not significantly ADP-ribosylated, and poly(ADP-Rib) polymerase activity was depressed two- to threefold. The data suggest that a subset of nucleosomes in extended regions of chromatin is subject to extensive ADP ribosylation.     

Monoclonal antibodies to poly(adenosine diphosphate ribose) recognize different structures

Two hybridomas producing monoclonal antibodies to poly(adenosine diphosphate ribose) [poly(ADP-Rib)] were established. One antibody, 10H (IgG3, kappa), bound to most of the poly(ADP-Rib) preparation, which consisted of molecules of various sizes of more than 20 ADP-Rib residues. The binding of this antibody was inhibited by not only poly-(ADP-Rib) but also a monomer unit of poly(ADP-Rib), Ado(P)-Rib-P. The sites protected by antibody 10H were isolated and analyzed by hydrolysis with alkaline phosphomonoesterase and then snake venom phosphodiesterase. The sites contained the same amounts of monomer units and branched portions [Ado(P)-Rib(P)-Rib-P] as the original poly(ADP-Rib) molecules but a lower average number of branched portions per molecule than in the original molecules. The other antibody, 16B (IgM, lambda), reacted with only 50% of the radioactive poly(ADP-Rib), and its binding was not inhibited by a monomer unit. This antibody protected 25% of all the poly(ADP-Rib) molecules from hydrolysis by snake venom phosphodiesterase. The protected sites contained twice as many branched portions per molecule as the original poly(ADP-Rib) molecules. These results show that the two monoclonal antibodies recognize different structures of poly-(ADP-Rib); 10H antibody recognizes the linear structure with ribose-ribose linkages, and 16B antibody may recognize specific structures, including the branched portions of poly-(ADP-Rib).     

Differentiation of a cell line of mouse myeloid leukemia : I. Simultaneous induction of lysosomal enzyme activities and phagocytosis

Induction of phagocytic activity in the Ml cell line of mouse myeloid leukemia, on being exposed to a conditioned medium from cultured embryo cells, was accompanied by an increment in the activities of both lysosomal acid phosphatase and acid protease. The activity of these lysosomal enzymes, as well as that of phagocytosis, was not induced when Ml cells were incubated either with the conditioned medium subjected to heat treatment or in the presence of 5-bromodeoxyuridine (BUdR). The levels of these induced enzyme activities in Ml cells were comparable to those in normal mouse peritoneal macrophages. The lysosomal enzyme activity in Mm-1 cells, which were spontaneously differentiated from Ml cells and exhibiting a higher phagocytic activity, were reminiscent of those in peritoneal macrophages. Based on these observations, it was concluded that both phagocytosis and lysosomal enzyme activity occur simultaneously during the course of differentiation. This differentiation, morphological or functional, in Ml cells in the presence of the conditioned medium was further supported by biochemical evidence.     

Activation of the Rap1 guanine nucleotide exchange gene, CalDAG-GEF I, in BXH-2 murine myeloid leukemia

Here we report the recurrent proviral activation of the Rap1-specific guanine nucleotide exchange factor CalDAG-GEF I (Kawasaki, H., Springett, G. M., Toki, S., Canales, J. J., Harlan, P., Blumenstiel, J. P., Chen, E. J., Bany, I. A., Mochizuki, N., Ashbacher, A., Matsuda, M., Housman, D. E., and Graybiel, A. M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13278-13283; Correction (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 318) gene in BXH-2 acute myeloid leukemia. We also show that CalDAG-GEF I encodes two protein isoforms, a full-length isoform (CalDAG-GEF Ia) and a C-terminally truncated isoform (CalDAG-GEF Ib). Expression of the full-length CalDAG-GEF Ia isoform in Rat2 fibroblasts enhances growth in low serum, whereas expression in Swiss 3T3 cells causes morphological transformation and increased saturation density. In FDCP1 myeloid cells, CalDAG-GEF Ia expression increases growth and saturation density in the presence of the diacylglycerol analogs phorbol 12-myristate 13-acetate (PMA), which activates CalDAG-GEF Ia exchange activity. Likewise, in 32Dcl3 myeloblast cells, CalDAG-GEF Ia expression increases cell adherence to fibronectin in response to PMA and calcium ionophore and allows higher saturation densities and prolonged growth on fibronectin-coated plates. These effects were correlated with increased Rap1, but not Ras, protein activation following PMA and calcium ionophore treatment. Our results suggest that Rap1-GTP delivers signals that favor progression through the cell cycle and morphological transformation. The identification of CalDAG-GEF I as a proto-oncogene in BXH-2 acute myeloid leukemia is the first evidence implicating Rap1 signaling in myeloid leukemia.     

Structure-Function Relationship of Three Triple-Helical Nucleic Acids

Abstract

The nucleic acid triplexes poly d(T)·poly d(A)·poly d(T), poly (U)·poly (A)·poly (U), and poly (I)·poly (A)·poly (I) display a sort of continuity between each other. However, their morphologies present their own individuality which, considering those of their parent duplexes, are quite unexpected. This comparison helps to understand triplex structure-function relationship. While helical parameters are functions of the sugar pucker, low values of WC and Hoogsteen base-pair propellers is commonplace for triplexes and the Hoogsteen base-pair geometry monitors the effects of the interstrand phosphates charge-charge repulsion.

Synopsis

The nucleic acid triplexes poly d(T)·poly d(A)·poly d(T), poly(U)·poly(A)·poly(U), and poly (I)·poly (A)·poly (I) present distinct morphologies. Considering those of their parent duplexes, they are also quite unexpected.     

Formation and characterization of antibody against 2''-(5"-phosphoribosyl)-5'' AMP, the monomer form of poly(adenosine diphosphate ribose).

Specific antibody against 2'-(5"-phosphoribosyl)-5'AMP (PR-AMP), a monomer of poly(adenosine diphosphate ribose) (poly(ADP-Rib)), was produced by immunizing a rabbit with PR-AMP coupled to bovine serum albumin (BSA). Antibody against PR-AMP was purified 53-fold from serum by (NH4) 2SO4 precipitation, and BSA-Sepharose 4B, DEAE-cellulose and (PR-AMP)-BSA-Sepharose 4B column chromatographies. Inhibition experiments show that the adenine ring, 5'-phosphate residue and ribose-ribose bond of PR-AMP were essential for the antigenic determinant of PR-AMP. Anti PR-AMP antibody bound, not only with PR-AMP, but also with poly(ADP-Rib) of various chain lengths, while anti poly(ADP-Rib) antibody bound with poly(ADP-Rib) but not with PR-AMP.     

Electrophoretic distribution and dissociation into subunits of lactate dehydrogenase derived from human myeloid leukemia cells before and after induction of differentiation

The electrophoretic distribution of lactate dehydrogenase (LDH) isoenzymes, Michaelis constant, reaction with substrate, and dissociation into subunits with guanidine hydrochloride was examined in undifferentiated and differentiated human myeloid leukemia cells. Differentiation was induced with 1/microgram/ml tunicamycin. Undifferentiated cells did not display phagocytic ability, and less than 5% of these cells had Fc receptors. After exposure to tunicamycin for 40 hr, 40% of these differentiated cells had Fc receptors, and 35% showed phagocytic activity after 160 hr. The majority of the LDH activity in the undifferentiated cells was found in fraction 3, and following differentiation almost a 50% reduction in LDH activity was observed in this fraction. In addition, LDH 3 isoenzyme levels were found to be greater in patients containing a high percentage of undifferentiated cells than in patients containing a high percentage of differentiated cells. Differentiated cells displayed LDH isoenzyme fraction pattern, Michaelis constant, and reaction with substrate similar to those found in the normal granulocytes. Differences in the dissociation of LDH into subunits with guanidine hydrochloride were found between undifferentiated and differentiated acute myeloid leukemia (AML) cells. Treatment with 0.75 M guanidine hydrochloride caused complete inactivation of LDH derived from normal differentiated cells, whereas similar treatment caused complete inactivation of LDH derived from AML or normal granulocytes. LDH isoenzymes derived from normal granulocytes and differentiated AML cells were also more sensitive to guanidine hydrochloride depression of fluorescence intensity. The sedimentation constant for single peak LDH at 5.5 M guanidine hydrochloride was calculated as 1.65 sec for differentiated and 1.70 sec for undifferentiated cells. The molecular weight of the polypeptide subunits for undifferentiated cells was 30,000 and for differentiated cells was 39,000. The apparent parallel between leukemic cells after induction of differentiation and normal granulocytes indicates that the leukemic cells retain their maturation potential when exposed to an inducer of differentiation.