Change in the activity of DNAse I inhibitor in the mouse spleen on the administration of a synthetic polyanion

Synthetic polyanion pyran (copolimer of divinyl ether and maleic anhydride) injected to mice increases the titre of antibody to sheep red blood cells as well as the activity of serum DNAase I and DNAase I splenic inhibitor. Simultaneously a growth of the spleen weight takes place. A possible role of the DNAase I inhibitor system in the mechanism of the adjuvant action of synthetic polyanion is discussed.     

Biological activity of calf spleen extracts containing a DNAase I inhibitor during irradiation

The purified calf spleen extract (I fraction CM-cellulose), possessing a highly active natural inhibitor of DNAase I, increases the survival rate of gamma-irradiated animals as opposed to irradiated controls. A possible role of DNAase I inhibitor in the radioprotective effect is discussed.     

Lysosomal cathepsins B, L, and D in the development of murine experimental leukemias

Lysosomal proteases are actively involved into pathogenesis of malignant tumors. Impairments in the interaction between proteases and their inhibitors are implicated in the processes of tumor invasion and metastasis. Among proteases associated with malignant growth, cysteine cathepsins B and L and aspartic cathepsin D are considered to play the major role in the tumor development. The present study was designed to investigate the activity of cathepsins B, L, and D during the development and treatment of murine experimental leukemias and to determine correlation between these proteases and course of pathological process as well as efficiency of the chemotherapeutic treatment. P-388 leukemia was characterized by a more aggressive development and unfavorable prognosis than L1210/1 leukemia. In mice with P-388 leukemia the activity of lysosomal cathepsins B, D, and L in the tumor tissue, liver and spleen, as well as the activity of cathepsins B and L in serum were lower than activities of these enzymes in mice with L1210/1 leukemia. Changes in the activity of cathepsins in liver and spleen of leukemic mice reflected a level of aggressiveness of the tumor development and invasion of these organs with tumor cells. Treatment of these experimental leukemias resulted in the increase of cathepsin B, L and D activity in the tumor tissue, liver, spleen and the increase in cathepsin B and L activity in serum. The highest protease activity was detected in the groups of mice characterized by the highest inhibition of the tumor growth. These data demonstrate that lysosomal proteases are involved in the progression of murine experimental leukemias and elimination of tumor cells in the result of treatment. Thus, determination of the activity of cysteine and aspartic proteases can be used for evaluation of cancer malignancy, tumor sensitivity for chemotherapy and efficiency of treatment.     

Inhibitor of cyclin-dependent kinase (CDK) interacting with cyclin A1 (INCA1) regulates proliferation and is repressed by oncogenic signaling

The cell cycle is driven by the kinase activity of cyclin·cyclin-dependent kinase (CDK) complexes, which is negatively regulated by CDK inhibitor proteins. Recently, we identified INCA1 as an interaction partner and a substrate of cyclin A1 in complex with CDK2. On a functional level, we identified a novel cyclin-binding site in the INCA1 protein. INCA1 inhibited CDK2 activity and cell proliferation. The inhibitory effects depended on the cyclin-interacting domain. Mitogenic and oncogenic signals suppressed INCA1 expression, whereas it was induced by cell cycle arrest. We established a deletional mouse model that showed increased CDK2 activity in spleen with altered spleen architecture in Inca1(-/-) mice. Inca1(-/-) embryonic fibroblasts showed an increase in the fraction of S-phase cells. Furthermore, blasts from acute lymphoid leukemia and acute myeloid leukemia patients expressed significantly reduced INCA1 levels highlighting its relevance for growth control in vivo. Taken together, this study identifies a novel CDK inhibitor with reduced expression in acute myeloid and lymphoid leukemia. The molecular events that control the cell cycle occur in a sequential process to ensure a tight regulation, which is important for the survival of a cell and includes the detection and repair of genetic damage and the prevention of uncontrolled cell division.     

A heat-stable protein inhibitor of cyclic 3',5'-nucleotide phosphodiesterase.

A heat-stable, non-dialyzable inhibitory factor of cyclic nucleotide phosphodieterase was detected in and partially purified from bovine retina. The factor appears to be a protein, since the inhibitory activity was abolished by trypsin digestion but not by DNAase or RNAase treatment. The protein inhibitor from bovine retina effectively inhibits the Ca2+-independent phosphodiesterase from several sources, including bovine retina, bovine rod outer segment, and a human lymphoblastic leukemia cell line, indicating lack of tissue and species specificity.     

A heat-stable protein inhibitor of cyclic 3′,5′-nucleotide phosphodiesterase

A heat-stable, non-dialyzable inhibitory factor of cyclic nucleotide phosphodiesterase was detected in and partially purified from bovine retina. The factor appears to be a protein, since the inhibitory activity was abolished by trypsin digestion but not by DNAase or RNAase treatment. The protein inhibitor from bovine retina effectively inhibits the Ca²⁺-independent phosphodiesterase from several sources, including bovine retina, bovine rod outer segment, and a human lymphoblastic leukemia cell line, indicating lack of tissue and species specificity.     

Effect of Ca2+, Mg2+-dependent deoxyribonuclease on DNA synthesis in cell nuclei from embryos of the sea urchin Strongylocentrotus intermedius

The sea urchin embryo nuclei which retained their ability to maintain the DNA synthesis in an in vitro system were isolated. The DNA synthesis isolated nuclei was shown to be an ATP-dependent process which is inhibited by low concentrations of actinomycin D, a polymerase alpha araCTP inhibitor. The newly synthesized DNA is represented by short fragments of about 4S. After addition of Ca2+, Mg2+-dependent DNAase to sea urchin embryo nuclei, the synthesis of short DNA fragments is enhanced. This stimulating effect of Ca2+, Mg2+-dependent DNAase is ATP-dependent and is observed only within a narrow range of enzyme concentrations (of the order of 1-5 units of DNAase activity per ml of incubation sample). The increase in the enzyme concentration to 10 or more units of activity results in the depression of DNA synthesis. It is concluded that DNA replication in sea urchin embryo nuclei depends on the presence of active DNAases as well as on the number of accessible initiation sites of DNA replication.     

The association of actin with a thyroid lysosomal fraction.

A lysosome-enriched fraction was prepared from bovine thyroid tissue using sucrose gradient centrifugation. An inhibitor of DNAase I was found to co-sediment and co-purify with the lysosomal fraction. This inhibitory activity is blocked by heavy meromyosin in the absence of ATP, and a component of 42000 molecular weight can be isolated by affinity chromatography on DNAase I linked to Sepharose. These results are consistent with the presence of an actin-like protein in a lysosome-enriched preparation from bovine thyroid tissue. Also, an increase in the level of membrane-associated actin is observed in response to thyrotropin stimulation of the thyroid tissue     

Effects of glucocorticoids on polyamine metabolism in liver and spleen of guinea pig during sensitization

Summary. Glucocorticoids are potent anti-inflammatory and immunosuppressive agents. As endogenous inhibitors of cytokine synthesis, glucocorticoids suppress immune activation and uncontrolled overproduction of cytokines, preventing tissue injury. Also, polyamine spermine is endogenous inhibitor of cytokine production (inhibiting IL-1, IL-6 and TNF synthesis). The idea of our work was to examine dexamethasone effects on the metabolism of polyamines, spermine, spermidine and putrescine and polyamine oxidase activity in liver and spleen during sensitization of guinea pigs. Sensitization was done by application of bovine serum albumin with addition of complete Freund’s adjuvant. Our results indicate that polyamine amounts and polyamine oxidase activity increase during immunogenesis in liver and spleen. Dexamethasone application to sensitized and unsensitized guinea pigs causes depletion of polyamines in liver and spleen. Dexamethasone decreases polyamine oxidase activity in liver and spleen of sensitized guinea pigs, increasing at the same time PAO activity in tissues of unsensitized animals.     

In vivo development of cytolytic T lymphocytes (CTL) to hapten-altered self: MIs-disparate cells facilitate the response by neutralizing IL 2 inhibitor

Inability to develop CTL in vivo to hapten-altered self can be attributed in part to an inhibitor of interleukin 2 (IL 2) that is present in the serum of normal mice. We have shown earlier that hapten-specific CTL can be generated in C3H mice (H-2k, MIsc) provided CBA/J (H-2k MIsd) spleen cells are injected simultaneously with hapten-modified syngeneic spleen cells into the hind foot paws. In efforts to determine whether serum levels of the inhibitor of IL 2 are altered as a consequence of this successful immunization method, we have compared the activity of the inhibitor in serum at intervals after the injection of syngeneic spleen cells, CBA spleen cells, or TNP-C3H spleen cells alone or together with CBA spleen cells, by using a murine IL 2-dependent, cloned cytotoxic T cell line, CT-6. The results indicate that inhibitor was neutralized optimally 48 to 72 hr after injection of TNP-C3H spleen cells mixed with CBA/J spleen cells. The order in which neutralization occurred was as follows: TNP-C3H cells + CBA/J cells greater than CBA cells greater than TNP-C3H cells greater than normal C3H spleen cells. Furthermore, supernatants from cultures of C3H lymph node cells stimulated in vivo with CBA/J cells also contained IL 2 activity. Thus, injection of CBA/J cells with TNP-modified syngeneic spleen cells produces IL 2 in vivo in sufficient quantity to neutralize the activity of the inhibitor as well as to facilitate the maturation of pre-CTL into hapten-altered self-specific CTL.     

Comparative characteristic of serpin--alpha-1 proteinase inhibitor in human and mice serum

Serpin alpha-1-proteinase inhibitor have been studied in human subjects and in mice of different lines as acute phase reactant and during tumor development. In humans, there was no difference of serpin activity between men and women. Increased activity was noted in men with acute trauma (acute phase reaction). Comparatively to male, in female mice of different lines decreased activity of serum alpha-1-proteinase inhibitor, was shown. There was no increase of alpha-1-proteinase inhibitor activity during inflammation induced by zymosan administration in mice. alpha-1-proteinase inhibitor belongs to acute phase reactants in humans but not in mice; for mice alpha-2-macroglobulin is a more typical acute phase reactant as compared to alpha-1-proteinase inhibitor. Murine tumor development (hepatoma HA-1, lymphosarcoma LS, Lewis lung adenocarcinoma) was followed by a decreased activity of serum alpha-1-proteinase inhibitor both in successfully treated and untreated groups. According to data of literature, similar dated were obtained in humans with tumors. It was suggested that changes of expressiln of alpha-1-proteinase inhibitor by tumors and its secretion were involved in decreased activity of alpha-1-proteinase inhibitor.     

Implications of a 5'-nucleotidase inhibitor in human leukemic cells for cellular aging and cancer

5'-Nucleotidase activity of normal human embryonic lung fibroblasts (IMR-90) was found to be inhibited by the homogenates of seven different cell lines originated from patients with different kinds of leukemia and of fresh lymphocytes from a patient with Sezary syndrome (circulating T-cell lymphoma). About 97% of the inhibiting activity was found in the soluble fraction of RPMI 8402 cells, a cell line originated from the lymphocytes of a patient with acute lymphocytic leukemia. This inhibiting activity was not destroyed by dialysis, heating at 56 degrees C for 30 min, nor digestion with RNAase or DNAase. About 85% of the inhibiting activity was destroyed by digestion with papain at 37 degrees C for 1 h and it was destroyed completely by heating at 100 degrees C for 30 min. When the heated (56 degrees C for 30 min) soluble fraction of RPMI 8402 cells was mixed with the homogenate of IMR-90 cells, it had no effect on the activities of alkaline, neutral or acid phosphatases, nor of N-acetyl-beta-D-glucosaminidase or cytochrome c oxidase of IMR-90 cells. Preincubating the mixed samples for 1, 20 and 45 min, respectively, before adding the substrate, the heated soluble fraction of RPMI 8402 cells did not increase the percentage of inhibition for 5'-nucleotidase of the homogenate of IMR-90 cells. No inhibition of other enzyme activities was observed under similar conditions. These data suggest that the inhibiting activity is due to a protein(s) that is not a protease. The inhibiting activity was found in a single peak after the soluble fraction was fractionated by Sephadex G-100 chromatography and sedimentation centrifugation. The molecular weight of the inhibitor was found to be approx. 35,000 by comparing its retention volume and sedimentation rate with those of proteins of known molecular weight. The present study suggest that the previously reported undetectability of 5'-nucleotidase in permanent cell lines could be due to the presence of a protein inhibitor for 5'-nucleotidase in these human leukemic cell lines. It also supports the hypothesis that the increased 5'-nucleotidase activity in normal senescent cells in vitro may be a control in cellular aging that is missing from leukemic cells in vitro.     

DNA-ase I activity in the DNA-ase I-inhibitor system

An increase in the DNase I activity was revealed in the serum of mice after intraperitoneal injection of different antigens. This reaction is related to the antigen nature and is dissimilar in mice of different strains. The increased activity of the inhibitor in the spleen of DBA/2 and BALB/c mice was discovered after intravenous injection of bovine DNase I. A potential role of serum DNase I in the system DNase I-inhibitor is discussed.     

Non-histone proteins in fractionated chromatin before and after DNAse II treatment

Chromatin from spleen cells of normal, non-immunized mice and from mice 3 days after immunization with human immunoglobulin G was fractionated at increasing salt concentrations into three fractions: 0.35 M NaCl-soluble, 2 M NaCl-soluble and a residual fraction, dissociated in 2 M NaCl/5 M urea. The residual fraction of chromatin, homogeneous by ultracentrifugation and containing only 25% of the total chromatin DNA, was associated with proteins strongly labeled with [3H]tryptophan, [3H]methionine and [3H]leucine. This fraction was more sensitive to DNAase II treatment than was native, non-fractionated chromatin and it contained approx. 40% Mg2+-soluble DNA sequences. The template activity of the residual fraction was 6--7-times higher than that of non-fractionated chromatin. Fraction A, characteristic for non-immunized spleen cells, was present in three chromatin fractions and after DNAase II treatment it remained only in the residual fraction, which suggests that this fraction is associated with genes non-transcribed in non-immunized mice. Fractions I and B1 were found mainly in the residual fraction, and only in smaller amounts in the 0.35 M NaCl-soluble fraction. After DNAase II treatment, fractions I and B1 in chromatin from immunized mice disappeared, which suggests that these fractions may be associated with active transcribed sequences during the immune reaction.     

Respiration-stimulated activation of DNAase I associated with mitochondria]

The influence of respiration and Ca++ transport in the liver mitochondria on the activation of DNAase I, associated with these organelles, was studied. It was shown that 96% of the total activity of this enzyme in mitochondria is in the latent state. Aeration of the mitochondrial suspension leads to a sharp increase in the enzyme activity. The activation of DNAase I is inhibited by EGTA addition (optimal pH 8.0), and stimulated in mitochondria, releasing Ca++. It is concluded that the activation of DNAase I is dependent on the state of cellular energetics. Participation of mitochondrial phospholypase A, activated by the Ca++ release from mitochondria during DNAase I activation is suggested.     

Identification and characterization of a soluble suppressor factor(s) in the serum of AKR mice bearing lymphocytic leukemia

Leukemia in AKR mice was found to be associated with the presence of a serum factor(s) termed AKR leukemic suppressor factor (AKR-LSF). Suppression was quantitated by measuring the inhibition of PHA-stimulated [³H]thymidine incorporation by normal AKR spleen cells at various dilutions of leukemic mouse serum (LMS). AKR-LSF activity was expressed as units per milliliter, which is the reciprocal of the LMS dilution that inhibited [³H]thymidine uptake by 50% with respect to fetal calf serum control cultures. The amount of activity in the serum directly correlated to the rate of tumor cell growth. Mice receiving 10⁷ BW5147 transplanted leukemia cells had 130 ± 12 units of AKR-LSF activity/ml of serum compared to 40 ± 8 units/ ml for mice with spontaneous leukemia. Normal mouse serum contained 33 ± 11 units/ml. The leukemic serum exhibited no strain specificity in either phytohemagglutinin or lipopolysaccharide assays, but was found to be twofold more inhibitory against mouse spleen cells than that against rat spleen cells. Human lymphocyte blastogenesis was not inhibited by the leukemic serum. LMS did not inhibit the growth of L929 fibroblasts or murine tumor cells in vitro. Further work is necessary to determine what role the suppressor factor may play in the regulation of antitumor cell immunity.     

The interaction of bovine pancreatic deoxyribonuclease I and skeletal muscle actin

The rate of exchange of actin-bound nucleotide is decreased by a factor of about 20 when actin is complexed with DNAase I without affecting the binding constant of calcium for actin. Binding constants of DNAase I to monomeric and filamentous actin were determined to be 5 X 10(8) M-1 and 1.2 X 10(4) M-1 respectively. The depolymerisation of F-actin by DNAase I appears to be due to a shift in the G-F equilibrium of actin by DNAase I. Inhibition of the DNA-degrading activity of DNAase I by G-actin is of the partially competitive type.     

Some aspects of the desoxyribonuclease activities of animal tissues

It has been found that many animal tissues contain "acid" desoxyribonucleases with pH optima near 5.2. A chemical method for the determination of this activity is described. The pancreatic desoxyribonuclease crystallized by Kunitz and shown to have a neutral pH optimum occurs in the pancreas together with the "acid" enzyme, but only the "neutral" enzyme occurs in the pancreatic juice. The ratio of "neutral" to "acid" DNAase activities in the pancreas is greater than 200, but in all other tissues examined there is no appreciable concentration of the neutral enzyme. It is concluded that neutral DNAase, like trypsin or lipase, has a digestive function. Some problems in the activation of the secretory enzyme in neutral pancreatic extracts are described. This activation can be interpreted in terms of a specific inhibitor or an inactive form of the enzyme. A comparison of the "acid" DNAase activities of different organs of the calf, horse, chicken, mouse, and rat indicates a possible connection between the DNAase concentration of a tissue and its capacity for proliferation or regeneration. However, the comparative DNAase activities of fetal and adult tissues do not support the view that DNAase function is limited to some simple role in the mechanics of cell division. Studies on the incorporation of glycine-N¹⁵ into the desoxypentose nucleic acids of avian red cells, and mouse liver, pancreas, and kidney show that the N¹⁵ uptake into the DNA of the chromosome is most rapid in tissues with high DNAase concentrations. No N¹⁵ incorporation is observed in the DNA of avian red cells, which have negligible concentrations of the enzyme. The analyses of tissues and nuclei isolated in non-aqueous media show that the bulk of the enzyme occurs in the cytoplasm of the cell, and that nuclear concentrations vary from tissue to tissue. A theory relating the DNAase activity of the cell to its over-all desoxypentose nucleotide metabolism is discussed. No evidence has been found for the presence of inhibitors of the "acid" DNAase in animal tissues.     

Natural killer cell suppression of Friend virus-induced preleukemic hemopoietic stem cells.

To determine whether hemopoietic cells infected with Friend polycythemia-inducing spleen focus-forming virus (SFFVp) are conserved or suppressed via natural surveillance in leukemia-resistant adult mice, we engrafted C57BL/6 recipients with isologous transgenic (donor origin marker) or natural killer (NK) cell-deficient B6 beige marrow cells exposed to SFFVp in vitro. Both groups of primary recipients were viremic and nonleukemic. Spleen cells from primary SFFVp-infected chimeras were engrafted into irradiated leukemia-susceptible secondary recipients to reveal dormant leukemia and grew as tumors of donor origin in 8 of 38 (21%) and 33 of 47 (70%) instances, respectively. Treatment of marrow donors and recipients with anti-asialo GM1 serum resulted in the depression of NK cell activity and the rapid development of dormant leukemia. We conclude that NK cells are an effective surveillance mechanism able to suppress SFFVp-induced preleukemic stem cells.