TRF1 is a critical trans-acting factor required for de novo telomere formation in human cells.

The duplex telomere repeat (TTAGGG)(n) is an essential cis-acting element of the mammalian telomere, and an exogenous telomere repeat can induce chromosome breakage and de novo telomere formation at the site of a break (telomere seeding). Telomere seeding requires the telomere repeat (TTAGGG)(n) more stringently than does an in vitro telomerase assay, suggesting that it reflects the activity of a critical trans-acting element of the functional telomere, in addition to telomerase. Furthermore, telomere seeding is induced at a frequency fluctuating widely among human cell lines, suggesting variation in the activity of this hypothetical factor among cells. In this study, we investigated the cellular factor(s) required for telomere formation using the frequency of telomere seeding as an index and identified TRF1, one of the telomere repeat binding proteins, as an essential trans-acting factor. The exogenous telomere repeat induces telomere formation at a frequency determined by the availability of TRF1, even in telomerase-negative cells. Our study shows clearly that TRF1 has a novel physiological significance distinct from its role as a regulator of telomere length in the endogenous chromosome. The possible role of TRF1 in cell aging and immortalization is discussed.

Identification and characterization of cell lines with a defect in a post-adsorption stage of Sendai virus-mediated membrane fusion.

In the early stage of infection, Sendai virus delivers its genome into the cytoplasm by fusing the viral envelope with the cell membrane. Although the adsorption of virus particles to cell surface receptors has been characterized in detail, the ensuing complex process that leads to the fusion between the lipid bilayers remains mostly obscure. In the present study, we identified and characterized cell lines with a defect in the Sendai virus-mediated membrane fusion, using fusion-mediated delivery of fragment A of diphtheria toxin as an index. These cells, persistently infected with the temperature-sensitive variant Sendai virus, had primary viral receptors indistinguishable in number and affinity from those of parental susceptible cells. However, they proved to be thoroughly defective in the Sendai virus-mediated membrane fusion. We also found that viral HN protein expressed in the defective cells was responsible for the interference with membrane fusion. These results suggested the presence of a previously uncharacterized, HN-dependent intermediate stage in the Sendai virus-mediated membrane fusion.

Gene delivery systems using the Sendai virus.

Fusogenic liposome (FL) is a delivery system that can transfer encapsulated materials into living cells directly through membrane fusion. FL is a promising approach for gene therapy because it can deliver various genetic materials much more efficiently than other non-viral vectors without damaging the cell. FL-mediated gene transfer consists of two independent membrane fusion phenomena; generation of a FL by fusing a Sendai virus (SV) particle with a simple liposome encapsulating DNA, and successive fusion of the FL with cell membrane. The former requires viral F protein but no other special molecule on the liposomal membrane, whereas the latter may require the receptor (sialic acid) and unidentified assistant molecule(s) on the cell membrane. Further analysis suggests that these assistant molecule(s), not the receptor, may control the fusion and govern the cell specificity of FL-mediated delivery. This review has described a detailed analysis of these fusion phenomena and discussed possible applications of FL-mediated gene delivery to human gene therapy.

Gene transfer vectors based on Sendai virus.

A gene delivery system is a fundamental technology used in human gene therapy. In order to treat patients suffering from incurable metabolic diseases, we must be able to deliver genes efficiently in situ and induce stable gene expression in non-dividing tissue cells. However, none of the current gene transfer systems (both viral and non-viral) satisfies this goal. In order to develop a novel gene delivery system that is free from the defects of existing gene transfer vectors, we analyzed natural biological phenomena that involve gene transfer and expression, and made artificial components that mimic the functioning of these systems. Our recent results shed light on three major aspects of gene transfer and expression: (1) the direct delivery of DNA into cytoplasm using fusogenic liposomes, (2) the transfer of DNA from cytoplasm to nucleus with a nuclear localization signal, and (3) the stabilization of DNA in the nucleus as an independent replicon. The possible development of a hybrid vector by combining these components is discussed.

Hemoglobin types, erythrocyte membrane skeleton and plasma iron concentration in calves with poikilocytosis.

The pathogenesis of poikilocytosis in calves was investigated. Nine calves 1 to 2 months old exhibiting poikilocytosis (poikilocytic calves), were examined. The proportion of poikilocytes in their blood was more than 70% in two calves (severely poikilocytic), while it averaged 30% in the remaining seven calves (moderately poikilocytic). All of the polikilocytic calves exhibited mild anemia. The concentration of plasma iron in 8 of them was 20-30% of that in normal calves, but it increased as the calves aged. The polikilocyte count in moderately poikilocytic calves decreased in proportion to the increase of the plasma iron concentration, though a high poikilocyte count was observed in a severely poikilocytic calf even after the plasma iron concentration reached the normal level. Column chromatography of a hemoglobin preparation from all the calves examined revealed 5 distinct peaks. Three peaks were embryonic, fetal and adult hemoglobins, respectively. The other peaks were tentatively designated Hb-2 and Hb-3, respectively. The amount of Hb-2 in the two severely poikilocytic calves was about three times that in the other calves including those moderately poikilocytic, and it decreased as the calves aged. The decrease of Hb-2 in one severely poikilocytic calf was related to a reduction of the proportion of poikilocytes in its peripheral blood. There was no significant difference in age-dependent changes of hemoglobin types between moderately poikilocytic calves and normal calves. Furthermore, SDS-PAGE analysis of red cell membrane revealed that protein 4.2 consisted of two polypeptides in the two severely poikilocytic, and three moderately poikilocytic calves, while it consisted of a single polypeptide in the remaining calves examined. These results indicated that the abnormalities of hemoglobin composition and protein 4.2 in the erythrocyte membrane observed in the severely poikilocytic calves might be responsible for the morphological changes in the erythrocytes, whereas iron deficiency was thought to be the predominant cause of poikilocytosis in the moderately poikilocytic calves.

Reduction in the level of Gal(alpha1,3)Gal in transgenic mice and pigs by the expression of an alpha(1,2)fucosyltransferase.

Hyperacute rejection of a porcine organ by higher primates is initiated by the binding of xenoreactive natural antibodies of the recipient to blood vessels in the graft leading to complement activation. The majority of these antibodies recognize the carbohydrate structure Gal(alphal,3)Gal (gal epitope) present on cells of pigs. It is possible that the removal or lowering of the number of gal epitopes on the graft endothelium could prevent hyperacute rejection. The Gal(alpha1,3) Gal structure is formed by the enzyme Galbeta1,4GlcNAc3-alpha-D-galactosyltransferase [alpha(1,3)GT; EC 2.4.1.51], which transfers a galactose molecule to terminal N-acetyllactosamine (N-lac) present on various glycoproteins and glycolipids. The N-lac structure might be utilized as an acceptor by other glycosyltransferases such as Galbeta1,4GlcNAc 6-alpha-D-sialyltransferase [alpha(2,6)ST], Galbeta1,4GlcNAc 3-alpha-D-Sialyltransferase [alpha(2,3)ST], or Galbeta 2-alpha-L-fucosyltransferase [alpha(1,2)FT; EC 2.4.1.691, etc. In this report we describe the competition between alpha(1,2)FT and alpha(1,3)GT in cells in culture and the generation of transgenic mice and transgenic pigs that express alpha(1,2)Fr leading to synthesis of Fucalpha,2Galbeta- (H antigen) and a concomitant decrease in the level of Gal(alpha1,3)Gal. As predicted, this resulted in reduced binding of xenoreactive natural antibodies to endothelial cells of transgenic mice and protection from complement mediated lysis.

Influence of the chemical nature of side chain at beta 108 of hemoglobin A on the modulation of the oxygen affinity by chloride ions. Low oxygen affinity variants of human hemoglobin expressed in transgenic pigs: hemoglobins Presbyterian and Yoshizuka.

Hemoglobin A (HbA) and two low oxygen affinity variants of HbA, HbPresbyterian and HbYoshizuka, were produced in transgenic pigs and purified to homogeneity by ion-exchange chromatography. These two variants contain either lysine (HbPresbyterian) or aspartic acid (HbYoshizuka) instead of the normal asparagine residue at position beta 108 in HbA. Transgenic pigs expressed these variants at a level up to 11% and were healthy. Both HbPresbyterian and HbYoshizuka exhibited low O2 affinity (P50 of 21.2 and 18.9, respectively, compared with control HbA value of 11.8 in 0.1 M NaCl, pH 7.5) and retained normal cooperativity with Hill coefficients of 2.9 and 2.5, respectively. HbPresbyterian exhibited Bohr effect comparable with HbA. In contrast, HbYoshizuka had a diminished response to changes in pH. Thus the structural basis of reduced O2 affinity of these variants appears to be distinct: the consequence of mutation at beta 108 is a function of the chemical nature of the side chain. This is further confirmed by the sensitivity of the O2 affinity of the variants to the presence of Cl-. The O2 affinity of HbYoshizuka is insensitive to changes in Cl- concentration, whereas the O2 affinity of HbPresbyterian exhibited a pronounced and dramatic chloride effect. In fact, P50 of HbPresbyterian was identical to that of HbA at very low Cl- concentrations, and the P50 increased to >40 at 0.5 M Cl-. The chloride effect was completely abolished when HbPresbyterian was stabilized at the 2,3-diphosphoglycerate pocket by interdimeric cross-linking. Molecular modeling studies demonstrate that in HbPresbyterian, Cl- can bridge the epsilon-amino group of Lys beta 108 with either the guanidino group of Arg beta 104 or the epsilon-amino group of Lys alpha 99, resulting in the stabilization of the "T" structure. The utility of these low O2 affinity hemoglobins as cell-free oxygen carriers is discussed.

An isologous porcine promoter permits high level expression of human hemoglobin in transgenic swine.

We describe isologous promoter replacement as an approach to permit high level expression of human hemoglobin in transgenic swine. We linked the human beta globin genomic coding region to the porcine beta globin promoter and used this fusion gene in an expression construct containing the human beta locus control region and the human alpha and epsilon genes to produce transgenic pigs. The highest level of expression was 24% human (32g/liter) and 30% human alpha/pig beta hybrid (40g/liter) hemoglobin in one transgenic pig. This pig was bred to a non-transgenic animal resulting in the transmission of high level human hemoglobin expression to 5 of 12 progeny.

Survival of mice inoculated with non-differentiating myeloid leukemia cells is prolonged by the injection of an inducer of cell differentiation with a sensitizer.

The effect of injection of an inducer and sensitizer on the survival times of syngeneic SL mice inoculated with resistant mouse myeloid leukemia cells (Ml) was examined. In vitro, the resistant Ml cells could not be induced to differentiate into mature macrophages and granulocytes by inducer (certain proteins, bacterial lipopolysaccharides, or glucocorticoids) alone, but could be induced to differentiate by treatment with both the inducer and a sensitizer (actinomycin D). In vivo, lipopolysaccharide alone scarcely affected the survival of SL mice inoculated with the resistant cells, but lipopolysaccharide plus actinomycin D significantly prolonged their survival. Administration of both lipopolysaccharide and actinomycin D also prolonged the survival of athymic nude mice inoculated with resistant Ml cells. These results suggest that prolongation of the survival of SL mice inoculated with resistant Ml cells is associated with the induction of differentiation of the cells.

Prolongation of survival time of mice inoculated with myeloid leukemia cells by inducers of normal differentiation.

Studies were made on the effects of inducers on the leukemogenicity of sensitive mouse myeloid leukemia cells (M1) that could be induced to undergo cell differentiation into mature granulocytes and macrophages in vitro by incubation with inducers (certain proteins, bacterial lipopolysaccharides, or glucocorticoids) and of resistant M1 cells that could not be induced to differentiate into mature cells. Inducers of cell differentiation significantly enhanced the survival times of mice inoculated with sensitive cells but scarcely affected the survival times of mice inoculated with resistant cells. Some mice inoculated with the sensitive cells and treated with lipopolysaccharide did not develop leukemia. The sensitive and resistant clone cells contained similar common tumor-related surface antigens. Treatment with lipopolysaccharide was also effective in athymic nude mice inoculated with the sensitive M1 cells. Lipopolysaccharide or glucocorticoid significantly stimulated differentiation of the sensitive cells cultured in a diffusion chamber in vivo but had little effect on differentiation of resistant cells. These results suggest the possibility of treating, with partial success, leukemia in vivo with differentiation inducers.

Actinomycin D restores in vivo sensitivity to differentiation induction of non-differentiating mouse myeloid leukemia cells.

Resistant mouse myeloid leukemia cells could not be induced to differentiate in vitro into mature macrophages and granulocytes by incubation with ascitic fluid or dexamethasone as inducer. Neither could endogenous inducers acting on resistant cells maintained in a diffusion chamber in syngeneic SL mice induce differentiation. However, when resistant cells were pretreated in vitro with low doses of actinomycin D they became sensitive to inducer in vitro or in vivo. The concentration of actinomycin D effective for this effect did not induce differentiation. The effect of actinomycin D was not due to inhibition of cell growth, since sensitivity was not observed when resistant cells were pretreated with the growth inhibitor 5-fluorodeoxyuridine. When resistant cells were kept in a diffusion chamber in mice injected with low doses of actinomycin D they showed significant differentiation, suggesting that the in vivo effect of actinomycin D is partly attributable to sensitization of the resistant cells to endogenous inducers.

Sensitization of resistant myeloid leukemia clone cells by anti-cancer drugs to factor-stimulating differentiation.

Studies were made on the effect of cancer chemotherapeutic drugs on in vitro differentiation of a clone (R4) of mouse myeloid leukemic cells (MI) that is resistant to inducers. Treatment of the cells with 50% ascitic fluid (an inducer) plus 0.25 microgram/ml of adriamycin or 0.3 microgram/ml of daunomycin induced phagocytic activity and suppressed cell growth, but had little effect on cell viability; treatment with ascitic fluid or the drugs alone had no effect. In combination with ascitic fluid, mitomycin-C, hydroxyurea, 5-fluorouracil, or bleomycin also induced phagocytic activity, but 6-mercaptopurine, amethopterin, or aminopterin did not. These drugs also induced other differentiation-associated properties, lysozyme activity, and locomotive activity. The present results indicate that some cancer chemotherapeutic drugs sensitive resistant leukemic cells to an inducer of cell differentiation.

Induction of differentiation of Rauscher virus-induced mouse myeloid leukemia cells with a factor(s) in ascitic fluid and inhibitors of nucleic acid and protein syntheses.

Cell line R453, established from a Rauscher virus-induced myeloid leukemia in a C57BL/6 mouse, was induced to differentiate in vitro into macrophages and granulocytes with ascitic fluids from animals bearing various ascites tumors or from mice treated with complete Freund's adjuvant, conditioned media from various cell lines, and glucocorticoid hormone. Differentiated R453 cells had a morphology similar to that of macrophages and granulocytes in normal hematopoietic organs, and they phagocytized small paricles such as latex particles, moved in soft agar showing locomotive activity, and had Fc and C3 receptors on the cell surface. This induction of differentiation of R453 cells was markedly enhanced by addition of inhibitors of RNA synthesis (actinomycin D, nogalamycin, or chromomycin A3), protein synthesis (puromycin or cycloheximide), or DNA synthesis (methotrexate, hydroxyurea, 5-fluorodeoxyuridine, or 1-beta-D-arabinofuranosylcytosine) in the presence of ascitic fluid. Of the inhibitors, actinomycin D was the most effective at a low concentration (5 ng/ml) in stimulating induction of differentiation of R453 cells. However, these inhibitors alone did not induce differentiation of R453 cells. The factor(s) in ascitic fluid that stimulates differentiation of R453 cells was heat labile, nondialyzable, and inactivated by trypsin.

Differentiation of mouse myeloid leukemia cells is inhibited by a factor from non-differentiating leukemia cells.

Mouse myeloid leukemia cells (MI) were induced to differentiate by a factor(s) (D-factor) in ascitic fluid. An inhibitory activity (I-activity) for the induction of differentiation was present in conditioned medium and lysate of MI cells resistant to the D-factor. The I-activity was non-dialyzable, heat-labile and protease-sensitive. Most of the activity was recovered in the fraction precipitated with 30-50% saturated ammonium sulfate. The fraction inhibited induction of phagocytic activity, migrating activity and morphological changes in MI cells, which are typical properties of differentiated MI cells. Low levels of I-activity were detected in conditioned medium or lysate of MI cells sensitive to the D-factor. The resistant MI cells were sensitized to the D-factor by treatment with a low concentration (5-10 ng/ml) of actinomycin D. The I-activity in conditioned medium of actinomycin D-treated resistant cells decreased with development of sensitivity to the D-factor. These results suggest that production of the I-activity in the resistant cells is closely associated with resistance of the MI cells to the D-factor.

Inhibition of RNA and protein syntheses makes non-differentiating mouse myeloid leukemia cells sensitive to a factor(s) stimulating differentiation.

Treatment with ascitic fluid from animals bearing various tumors, can induce mouse myeloid leukemia line cells, M1, to differentiate in vitro into macrophages and granulocytes. Cells were isolated that were resistant to the ascitic fluid factor(s) stimulating differentiation (D-factor). The resistant cells became sensitive to the D-factor and differentiated after treatment with various inhibitors of RNA synthesis (actinomycin D, nogalamycin, chromomycin A3 or cordycepin) or protein synthesis (puromycin or cycloheximide). The cells could not be induced to differentiate by treatment with the inhibitors alone. The effective doses of the inhibitors of protein synthesis were toxic to the cells. Among these inhibitors actinomycin D (5 ng/ml) was the most effective for sensitizing the resistant cells. Inhibitors of DNA synthesis did not sensitize the resistant cells. Added actinomcyin D was mainly recovered in the nuclear fraction of the cells. The sensitizing effect of actinomycin D on the cells was roughly parallel to the extent of its inhibition of RNA synthesis in the cells. The effective concentration of actinomycin D (5 ng/ml) mainly inhibited it also inhibited alpha-amanitin-resistant RNA synthesis to some extent. These results suggest that alpha-amanitin-sensitive RNA synthesis may be involved in sensitization of the resistant cells to the D-factor.