Inhibition of invasive properties of murine melanoma by bovine pancreatic DNase I in vitro and in vivo

After a long pause, the accumulation of data on the involvement of tumor-specific DNA and extracellular DNA in metastasis has again placed enzymes with deoxyribonuclease activity in the focus of the search for antitumor and antimetastatic drugs. In this work, the ability of bovine pancreatic DNase I to reduce the invasive potential of B16 melanoma has been investigated in vitro and in vivo. It was found that DNase I had a cytotoxic effect on B16 melanoma cells (IC₅₀ ≈ 10⁴ U/mL). At the same time, significantly lower doses of DNase I (10²–10³ U/mL) inhibited the migratory activity of melanoma cells in vitro, causing a decrease in the distance of cell front migration and in the area of scratch healing 48 h after the enzyme addition, as well as reducing the rate of cell migration. In mice with B16 metastatic melanoma, intramuscular administration of DNase I in the dose range of 0.12–1.20 mg/kg resulted in a two-to threefold decrease in the number of surface lung metastases and caused nonspecific antigenic immune stimulation.     

Electrochemical assay for deoxyribonuclease I activity

A thiolated oligonucleotide having three ferrocenes was immobilized on a gold electrode through the sulfur-gold linkage. This electrode showed a current response based on the redox reaction of the ferrocene moieties and this response was decreased after treatment with deoxyribonuclease I (DNase I), suggesting the disappearance of the ferrocene moieties on the electrode by the DNase I digestion. A linear correlation between i₀ and i, which are current peaks before and after DNase I treatment, respectively, was observed and this slope was decreased with increase in the amount of DNase I. No current decrease was observed in the presence of EDTA or RNase A instead of DNase I. These results suggested that the current decrease responded specifically to the amount of DNase I and this electrode could be used for an electrochemical DNase I assay. Under the optimum conditions of DNase I digestion at 37 °C for 30 min, a quantitative analysis could be achieved in the range of 10⁻⁴-10⁻² units/μl of DNase I.     

Immunization of rabbits with DNase I produces polyclonal antibodies with DNase and RNase activities

Immunization of rabbits with DNase I leads to the production of antiidiotypic Abs with DNase activity. It is not known at present whether antiidiotypic Abs against DNA-hydrolyzing enzymes can possess RNase activity. Here we show that immunization of healthy rabbits with bovine DNase I produces IgGs with intrinsic DNase and RNase activities. Electrophoretically and immunologically homogeneous polyclonal IgGs were obtained by sequential chromatography of the immune sera on Protein A-Sepharose and gel filtration. Affinity chromatography on DNA cellulose using elution of Abs with different concentrations of NaCl and an acidic buffer separated catalytic IgGs into four Ab subfractions, three of which demonstrated only DNase activity while one subfraction hydrolyzed RNA faster than DNA. The serum of patients with many different autoimmune (AI) diseases contains small fractions of antibodies (Abs) interacting with immobilized DNA, which possess both DNase and RNase activities. Our data suggest that a fraction of abzymes from AI patients hydrolyzing both DNA and RNA can contain a subfraction of Abs against DNase I.     

Antibodies against RNA hydrolyze RNA and DNA

Immunization of animals with DNA leads to the production of anti-DNA antibodies (Abs) demonstrating both DNase and RNase activities. It is currently not known whether anti-RNA Abs can possess nuclease activities. In an attempt to address this question, we have shown that immunization of three rabbits with complex of RNA with methylated BSA (mBSA) stimulates production of IgGs with RNase and DNase activities belonging to IgGs, while polyclonal Abs from three non-immunized rabbits and three animals immunized with mBSA are catalytically inactive. Affinity chromatography of IgGs from the sera of autoimmune (AI) patients on DNA-cellulose usually demonstrates a number of fractions, all of which effectively hydrolyze both DNA and RNA, while rabbit catalytic IgGs were separated into Ab subfractions, some of which demonstrated only DNase activity, while others hydrolyzed RNA faster than DNA. The enzymic properties of the RNase and DNase IgGs from rabbits immunized with RNA distinguish them from all known canonical RNases and DNases and DNA- and RNA-hydrolyzing abzymes (Abzs) from patients with different AI diseases. In contrast to RNases and AI RNA-hydrolyzing Abs, rabbit RNase IgGs catalyze only the first step of the hydrolysis reaction but cannot hydrolyze the formed terminal 2',3'-cyclophosphate. The data indicate that Abzs of AI patients hydrolyzing nucleic acids in part may be Abs against RNA and its complexes with proteins. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Effect of neurectomy on nuclease activity in skeletal muscles of rats

Author followed up the activity of the three enzymes involved in the catabolism of nucleic acids--acid deoxyribonulease (DNase II), alkaline ribonuclease (RNase I), and acid ribonuclease (RNase II)--in the denervated gastrocnemius and soleus muscles of rats for 28 postoperative days. The activity of both acid nucleases increased in both types of denervated muscles, compared with the respective controls. Up to the 14th postoperative day, the activity excess of both acid nucleases was more significant in the m. gastrocnemius than in the m. soleus. The RNase I ran below the control activity during the whole period in the m. soleus and up to the 14th day in the m. gastrocnemius. The role of nucleases and nuclease inhibitors in the changes of nucleic acid catabolism in neurogenic muscular atrophies is discussed.     

Ultrastructural Cytochemistry of Secretory Granules of Esophageal Glands of Meloidogyne incognita

Ultrastructural cytochemical tests for several enzymes, proteins, carbohydrates, and nucleic acids were conducted on secretory granules o£ dorsal and subventral esophageal glands of preparasitic second-stage juveniles and the dorsal gland of adult females of Meloidogyne incognita. Secretory granules in the subventral glands of juveniles stained positive for acid phosphatase. Peroxidase, DNase, RNase, cellulase, and nucleic acids were not detected in these granules. Secretory granules in the dorsal gland of adult females stained positive for peroxidase (pH 7.6) in < 50% of the tests, Acid phosphatase, β-glucuronidase, DNase, RNase, polyphenoloxidase, cellulase, and carbohydrates were not detected in dorsal gland granules in adult females. Positive staining with cobalt thiocyanate, a stain for amino groups of basic proteins, occurred in secretory granules in the dorsal gland, ribosomes, and chromatin in adult females. Ribosomes, nuclei, and secretory granules of the dorsal gland of adult females intensely stained when incubated in three reagents specific for nucleic acid.     

Calcium protects DNase I from proteinase K: a new method for the removal of contaminating RNase from DNase I

DNase I and proteinase K are two enzymes commonly used in the purification of highly polymerized RNA. In the presence of EDTA DNase I is rapidly inactivated by proteinase K while in 10 mm Ca²⁺ DNase is totally immune to proteinase K inactivation even at protease concentrations of up to 1 mg/ml. RNase A, a common contaminant of “RNase-free” DNase was inactivated by proteinase K in the presence or absence of Ca²⁺. Treatment of DNase I with proteinase K in the presence of Ca²⁺ selectively removed RNase A activity as judged by rRNA and poly(A⁺ RNA ribosomal RNA degradation monitored by sucrose gradient centrifugation. These results suggest that (i) DNase A and proteinase K can be used together in the presence of Ca²⁺ to obtain better digestion of nucleoprotein complexes, and (ii) proteinase K treatment of Ca²⁺ DNase can be used to selectively remove contaminating RNase.     

A novel glycoprotein obtained from Chlorella vulgaris strain CK22 shows antimetastatic immunopotentiation

 A glycoprotein extract (CVS), derived from the unicellular green alga Chlorella vulgaris, strain CK22, exhibited a pronounced antitumor effect against both spontaneous and experimentally induced metastasis in mice. Inhibition of tumor metastasis was enhanced when intratumor administration of CVS was followed by s.c. injection of CVS. Anti-metastatic immunopotentiation was observed in euthymic mice, but not in athymic nude mice. The antitumor activity of CVS was reflected in antigen-specific, T-cell-mediated immunity. Both CD4 and CD8 T cells contributed to the antimetastatic effects, as shown by in vivo depletion experiments with anti-T-cell subset antibodies. Furthermore, CVS caused the recruitment of T cells to the regional lymph nodes and their proliferation in these organs. The CD4-positive population, following CVS injection at the time of tumor rechallenge, displayed a pronounced increase in the proportion of T cells that were CD18 bright, CD44 bright, CD25⁺, CD54⁺, CD69⁺ or CD71⁺ in the lymph nodes. Thus, CVS induces T cell activation in peripheral lymph nodes in tumor-bearing mice. We conclude that CVS augments antimetastatic immunity through T cell activation in lymphoid organs and enhances recruitment of these cells to the tumor sites. Presurgical treatment with CVS might prevent metastasis or tumor progression. Received: 5 June 1997 / Accepted: 12 September 1997     

Role of histamine in natural killer cell-mediated resistance against tumor cells

The formation of lung metastases by i.v.-injected B16 melanoma (F1 and F10 strain) cells in Swiss albino, C57BL/6, and BALB/c mice was reduced by a single dose of histamine given 24 h before tumor cell inoculation. The antimetastatic effect of histamine was specifically mediated by histamine H2-receptors (H2R): it was blocked by the H2R antagonist ranitidine and mimicked by dimaprit, a specific H2R agonist but not by an H2R-inactive structural analog of this compound, nor-dimaprit, or the H1R agonist 2-thiazolyl-ethylamide. A single dose of any of the H2R antagonists ranitidine, tiotidine, famotidine, or cimetidine drastically augmented metastasis. Effects of H2R-interactive compounds on B16 metastasis required intact NK cells, as judged by the inability of histamine or ranitidine to affect B16 metastasis after NK cell depletion in vivo using antibodies to asialo-GM1. NK-cell-mediated lysis of YAC-1 lymphoma cells in vivo was enhanced by histamine and reduced by ranitidine within 4 h after inoculation of tumor cells. The antimetastatic effect of IL-2 was potentiated by histamine; in some experiments, combined treatment with a low dose of IL-2 (6000 U/kg) and histamine completely eliminated metastasis, whereas concomitant treatment with ranitidine abrogated antimetastatic effects of IL-2; animals treated with ranitidine and IL-2 displayed the same level of enhanced metastasis as those treated with ranitidine alone. The presented data are suggestive of an earlier unrecognized role for histamine in NK cell-mediated resistance against metastatic tumor cells.     

Characteristics of stimulation of gonadotropin secretion by kisspeptin-10 in female goats

The aims of the present study were to clarify the effect of kisspeptin-10 (Kp10) on the secretion of luteinizing hormone (LH), follicle stimulating hormone (FSH), growth hormone (GH) and prolactin (PRL) in goats, and compare the characteristics of any response with those of the response to gonadotropin-releasing hormone (GnRH). The experiments were performed using four female goats (4–5 years old) in the luteal phase of estrous cycle. A single intravenous (i.v.) injection of 1, 5 and 10 μg/kg b.w. (0.77, 3.85 and 7.69 nmol/kg b.w.) of Kp10 stimulated the release of LH. Maximum values were observed 20–30 min after the injection. On the other hand, Kp10 did not alter plasma GH and PRL concentrations significantly. Three consecutive i.v. injections of Kp10 (5 μg/kg b.w.) or GnRH (5 μg/kg b.w.: 4.23 nmol/kg b.w.) at 2-h intervals increased both plasma LH and FSH levels after each injection (P < 0.05); however, the responses to Kp10 were different from a similar level of GnRH. The rate of decrease in LH and FSH levels following the peak was attenuated in Kp10-treated compared to GnRH-treated animals. These results show that Kp10 can stimulate the release of LH and FSH but not GH and PRL in female goats and suggest that the LH- and FSH-releasing effect of the i.v. injection of Kp10 is less potent than that of GnRH.     

Rifamycin derivatives: specific inhibitors of nucleic acid polymerases

Rifampicin and three rifamycin SV derivatives with different lipophilic side chains were tested as inhibitors of a number of purified enzymes including the α and αβ forms of RNA-directed DNA polymerase of avian myeloblastosis virus (AMV). $\text{AF}\text{ABDMP}$ (2,5-dimethyl-4-N-benzyl demethyl rifampicin), $\text{AF}\text{013}$ (O-n-octyloxime of 3-formyl rifamycin SV) and C-27 (rifamycin SV with a dicyclohexylalkyl substituted piperidyl ring at the 3-position) at concentrations less than 20 to 40 μg/ml completely inhibited the RNA- and DNA-directed DNA polymerase and RNase H activities of both AMV enzymes. Rifampicin was inactive at 100 μg/ml. When used against a variety of non-polymerizing enzymes such as alkaline phosphatase, glutamate-oxaloacetate transaminase, DNase I, and RNase A, these derivatives were inactive at drug concentrations between 100 and 200 μg/ml. Polynucleotide phosphorylase was inhibited slightly by all three derivatives. These results support the idea that rifamycin SV derivatives with appropriate 3-substituted side-chains are specific inhibitors of nucleic acid polymerizing enzymes.     

An efficient method to eliminate the protease activity contaminating commercial bovine pancreatic DNase I

A method was developed to eliminate the proteases contaminating commercial DNase I, which can cause degradation of target protein during the purification process. Bio Basic DNase stock solution (in Tris–HCl buffer [pH 8.0] containing 5 mM CaCl₂) was first incubated at 50 °C to generate autolysis of proteases and zymogens, leading to a significant reduction in protease activity while preserving DNase activity. The residual protease activity was completely inhibited by further incubation with 2 mM PMSF (phenylmethylsulfonyl fluoride) or 2× S8830 inhibitor cocktail. This approach could be readily applicable to eliminate the protease activity in any DNase products or during the preparation of commercial DNase.     

Effect of amphotericin B and its combination with 5-fluorouracil on the development of transplantable rat lymphosarcoma]

The studies were carried out on rats with subcutaneously transplanted metastizing Pliss lymphosarcoma. The animals were treated with amphotericin B administered intravenously in complex with sodium desoxycholate in doses of 336 and 20 Units/kg for 7 days beginning from 24th hour after the tumor transplantation. The treatment resulted in a statistically reliable inhibition of the tumor metastasizing as compared to the control animals. When the animals with the tumors were subjected to combined therapy with 5-fluoruracyl and amphotericin B, the antitumor and antimetastatic effect of 5-fluoruracyl significantly increased. Determination of amphotericin B binding with the organ homogenates showed that the antibiotic bound with the homogenates or the organs of the rats with the tumors to a greater extent than with those of the intact animals. The levels of the antibiotics in the tumor metastases were higher than those in the primary tumors.     

Picropodophyllin inhibits tumor growth of human nasopharyngeal carcinoma in a mouse model

Insulin-like growth factor-1 receptor (IGF-1R) is a cell membrane receptor with tyrosine kinase activity and plays important roles in cell transformation, tumor growth, tumor invasion, and metastasis. Picropodophyllin (PPP) is a selective IGF-1R inhibitor and shows promising antitumor effects for several human cancers. However, its antitumor effects in nasopharyngeal carcinoma (NPC) remain unclear. The purpose of this study is to investigate the antitumor activity of PPP in NPC using in vitro cell culture and in vivo animal model. We found that PPP dose-dependently decreased the IGF-induced phosphorylation and activity of IGF-1R and consequently reduced the phosphorylation of Akt, one downstream target of IGF-1R. In addition, PPP inhibited NPC cell proliferation in vitro. The half maximal inhibitory concentration (IC50) of PPP for NPC cell line CNE-2 was ?1 μM at 24 h after treatment and ?0.5 μM at 48 h after treatment, respectively. Moreover, administration of PPP by intraperitoneal injection significantly suppressed the tumor growth of xenografted NPC in nude mice. Taken together, these results suggest targeting IGF-1R by PPP may represent a new strategy for treatment of NPCs with positive IGF-1R expression.     

Application of general ligand affinity chromatography for the mutual separation of deoxyribonuclease and ribonuclease free of protease contamination

α-Chymotrypsin and trypsin could be removed from mixtures of pancreatic RNase and DNase I by chromatography on the general ligand affinity column of 8-(6-aminohexyl)-amino-5′-AMP-Sepharose. Both RNase and DNase are quantitatively recovered by elution with 5′-AMP. The separation of RNase and DNase from each other was carried out by the specific retention of RNase on 8-(6-aminohexyl)-amino-2′-AMP-Sepharose at high ionic strength (0.1 m); DNase failed to adsorb under these conditions. RNase was then desorbed in the presence of a mononucleotide. This is a facile method for obtaining pure enzymes from commercial preparations and gives rise to the potential application of these columns for the analysis of enzyme-nucleotide interactions.     

Anti-CD40 antibody induces antitumor and antimetastatic effects: the role of NK cells

We assessed the effect of the stimulatory anti-CD40 Ab on NK cell activation in vivo and the therapeutic potential of activated NK cells in tumor-bearing mice. Single-dose i.p. injection of the anti-CD40 Ab resulted in production of IL-12 and IFN-gamma in vivo, followed by a dramatic increase in NK cell cytolytic activity in PBLs. NK cell activation by anti-CD40 Ab was also observed in CD40 ligand knockout mice. Because NK cells express CD40 ligand but not CD40, our results suggest that NK activation is mediated by increased cytokine production upon CD40 ligation of APCs. Treatment of tumor-bearing mice with anti-CD40 Ab resulted in substantial antitumor and antimetastatic effects in three tumor models. Depletion of NK cells with anti-asialo GM1 Ab reduced or abrogated the observed antitumor effects in all the tested models. These results indicate that a stimulatory CD40 Ab indirectly activates NK cells, which can produce significant antitumor and antimetastatic effects.     

Destabilizing mutations alter the hydrogen exchange mechanism in ribonuclease A

The effect of strongly destabilizing mutations, I106A and V108G of Ribonuclease A (RNase A), on its structure and stability has been determined by NMR. The solution structures of these variants are essentially equivalent to RNase A. The exchange rates of the most protected amide protons in RNase A (35°C), the I106A variant (35°C), and the V108G variant (10°C) yield stability values of 9.9, 6.0, and 6.8 kcal/mol, respectively, when analyzed assuming an EX2 exchange mechanism. Thus, the destabilization induced by these mutations is propagated throughout the protein. Simulation of RNase A hydrogen exchange indicates that the most protected protons in RNase A and the V108G variant exchange via the EX2 regime, whereas those of I106A exchange through a mixed EX1 + EX2 process. It is striking that a single point mutation can alter the overall exchange mechanism. Thus, destabilizing mutations joins high temperatures, high pH and the presence of denaturating agents as a factor that induces EX1 exchange in proteins. The calculations also indicate a shift from the EX2 to the EX1 mechanism for less protected groups within the same protein. This should be borne in mind when interpreting exchange data as a measure of local stability in less protected regions.     

Therapeutic efficacy of tumor-targeted IL2 in LTα−/− mice depends on conditioned T cells

An effective immunological eradication of tumors by the adaptive immune system depends on T cell priming, expansion of specific T cells and their effector function. It has been shown that either step may be impaired in the tumor-bearing host, and several strategies have been used to improve antitumor immune responses. In this regard, tumor-targeted IL2 therapy leads to the destruction of established melanoma metastases in fully immune competent mice as previously demonstrated. This effect has been attributed, but never directly confirmed, to the boost of antigen-experienced T cells. To this end, we demonstrate the absence of any antitumor effect of targeted IL2 in mice characterized by an impaired priming of T cell responses. Notably, in these animals tumor-targeted IL2 therapy induced tumor regression only after adoptive transfer of tumor-conditioned splenocytes. A detailed analysis revealed that T cells present within the transferred splenocytes were actively participating in the immune response as these were clonally expanded after targeted IL2 therapy. In summary, we demonstrate here that in LTα^−/− mice lacking sufficient numbers of tumor-specific T cells only the passive transfer of such cells prior to therapy restores the efficacy of tumor-targeted IL2 therapy. Thus, the antitumor effect of tumor-targeted IL2 is indeed based on the boost of pre-existing T cell responses.