Lipoplexes prepared from cationic liposomes and mammalian DNA induce CpG-independent, direct cytotoxic effects in cell cultures and in mice

BACKGROUND: Recent studies demonstrated the cytotoxic activity of bacterial DNA (pDNA) complexed with cationic lipids. This cytotoxicity is related to the ability of pDNA to induce potently the immune system, which is associated with release of inflammatory cytokines. Both activities seem to be related to the nonmethylated CpG sequences present in the pDNA. Here we study the cytotoxic activity of nonbacterial DNA complexed with cationic lipids against various tumor cell lines. METHODS: Various nucleic acids complexed with cationic liposomes were prepared and their cytotoxic activity was studied in cell cultures and in tumor-bearing mice. Cell uptake of lipoplexes was evaluated, and mechanism of DNA cytotoxic activity was studied. RESULTS: We found that nonbacterial (vertebrate) genomic DNA when complexed with cationic lipids is highly cytotoxic against C-26 and M-109 tumor cells. Cationic lipids alone were not toxic to these cells. The cytotoxic activity does not result from nonspecific acidification of the intracellular milieu, as substitution of DNA by poly-L-glutamate did not result in cytotoxicity, although the level of uptake of anionic charges per cell was similar to that of the nucleic acids, suggesting that this cytotoxic effect is specific to nucleic acids. By studying the nucleic acid fate using confocal microscopy, we found that cytotoxicity correlated with the release of DNA into the cytoplasm following uptake of lipoplexes. Injection of calf thymus DNA-based lipoplexes to mice with peritoneal C-26 metastases resulted in doubling of median survival time and long-term survival in 20% of the tumor-bearing mice. Judging by low levels of IFN-gamma, TNF-alpha and IL-6 in the treated mice, this effect cannot be ascribed to Th-1 inflammation, but rather to a direct cytotoxic effect on the tumor cells. CONCLUSIONS: The above data provide a new insight into the mechanisms of lipoplex-mediated antitumor effects in vitro and in vivo and new perspectives in cancer therapy.     

Differential inhibition of DNA and RNA biosynthesis in HeLa S3 cells by tetaine, a dipeptide antibiotic

A dipeptide antibiotic, tetaine, was found to diminish the rate of incorporation of 3H-labelled precursors into nucleic acids of intact and permeabilized HeLa S3 cells with concomitant negligible effect on protein synthesis. Comparison of the inhibitory effects of tetaine indicates that the antibiotic at 0.03-0.1 mM is a selective inhibitor of cellular DNA biosynthesis and, at higher concentration, of DNA and RNA biosynthesis. Tetaine is also an inhibitor of DNA and RNA polymerase reactions in a cell-free system, as determined using partially purified extracts from HeLa S3 cells that served as a source of the enzymes. The pretreatment experiments showed that tetaine inactivated the polymerases without affecting DNA template function. The tetaine effect on biosynthesis of nucleic acids in HeLa S3 cells can be attributed rather to the intact antibiotic than to the product of its enzymatic cleavage, anticapsin.     

Reagents specific for cell surface components.

Mercury, diazonium ions and dyes which bind nucleic acids were covalently linked to dextrans using methods that resulted in non-hydrolyzable reagent-dextran bonds without impairing the binding abilities of the reagents, i.e. these dextran derivatives reacted with thiols, phenols/imidazoles and nucleic acids respectively. Since these dextran derivatives cannot penetrate into cells and since dextran itself does not bind to cells, these compounds represent reagents specific for the cell surface. They may be used both to evaluate cell surface constituents of intact cells and to affect viable cells via an interaction with those constituents. Mercury-dextran was found to bind to cells; the amount of mercury thus attached to the cells was about ten times smaller than when an equivalent concentration of free mercury ions was used. Mercury-dextran, bound to cells after a 30-min exposure at room temperature, was localized on the surface of these cells, as sodium borohydride reduced this complex giving rise to the intact cells, elementary mercury and free dextran which was released into medium. When cells were constantly exposed to the mercury-dextran, its toxic effects were comparable to that of free mercury ions. Diazonium-dextran, which also binds tightly to the cell surface, was also considerably toxic. Dextrans substituted with dyes which bind to nucleic acids were less toxic than the parent dyes themselves; it was shown that the attachment of such a dye to dextran decreased the binding of dye to cells under detection limits.     

Effects of the trivalent and hexavalent chromium on the physicochemical properties of mammalian cell nucleic acids and synthetic polynucleotides

The effects of trivalent (chromium chloride) and hexavalent (potassium dichromate) chromium have been studied on the nucleic acids of cultured mammalian cells (BHK hamster fibroblast line), commercial DNA and RNA, and synthetic polynucleotides of known base composition. Modifications of UV absorption spectra and alterations of thermal denaturation and renaturation patterns have been observed by directly treating purified nucleic acids, as well as by examining nucleic acids extracted from cells treated with chromium compounds.Cr(III) interacts with nucleic acid bases, mostly guanine and cytosine, but also with phosphate groups, leading to deprotonation of bases as well as intramolecular cross-links, sandwich complexes between bases and chelation between bases and phosphates. Such interactions destabilize the DNA structure. On the contrary, stabilization of RNA, due to intramolecular metal bonds between nitrogen bases in GC-rich regions, is mainly produced. The kind of interaction of Cr(III) with nucleic acids is not significantly different when intact BHK cells are treated.Cr(VI) interacts similarly with DNA and RNA giving instead different effects when purified nucleic acids or intact cells are treated. Treatment of purified DNA produces breakages in the polynucleotide chain due to the oxidizing power of Cr(VI). In intact cell treatments, changes in the properties of DNA are observed. These could result from the combined action of Cr(III), produced by the intracellular reduction of Cr(VI) and the oxidizing activity of residual Cr(VI).The relevance of Cr(VI) and Cr(III) interactions to the mechanisms of chromium (carcino)genic action is summarized. It is stressed that Cr(VI), if not completely reduced to Cr(III) by extracellular and endoplasmic constituents, can reach the cell nucleus and directly interact with DNA.     

Nucleic Acid Scavenging Polymers Inhibit Extracellular DNA-Mediated Innate Immune Activation without Inhibiting Anti-Viral Responses

Toll-like receptor (TLR) family members, 3, 7 and 9 are key components in initiation and progression of autoimmune disorders such as systemic lupus erythematosus (SLE). These TLRs are often referred to as nucleic acid-sensing TLRs based on their ability to recognize DNAs or RNAs produced by pathogens or damaged cells. During autoimmune disease progression these receptors recognize self nucleic acids as well as self nucleic acid-containing complexes and contribute to inflammatory cytokine production and subsequent enhancement of serum autoantibody levels. We have recently discovered that nucleic-acid scavenging polymers (NASPs) can neutralize the proinflammatory effects of nucleic acids. Here, we begin to explore what effects such NASPs have on normal immune function. We show that such NASPs can inhibit TLR activation without affecting nucleic acid-independent T cell activation. Moreover, we observe that stimulation of immune cells by encapsulated nucleic acids, such as those found in viral particles, is unaffected by NASPs. Thus NASPs only limit the activation of the immune system by accessible extra-cellular nucleic acid and do not engender non-specific immune suppression. These important findings suggest that NASPs represent a new approach toward anti-inflammatory drug development as these agents can potentially be utilized to block overt autoimmune disorders and inflammation while allowing normal immune responses to occur.     

Interaction of antitumor drugs with human erythrocyte ghost membranes and mastocytoma P815: a spin label study.

The cytotoxic and mutagenic properties of antitumor drugs such as adriamycin, acridines, diacridine, actinomycin D and Pt compounds are related to their interaction with nucleic acids and inhibition of protein synthesis. We have examined their interaction with human erythrocyte ghost membranes and murine mastocytoma cells using spin labeling techniques. These drugs induce changes in electron spin resonance of the spin labeled ghost membranes and in the mastocytoma cells. These alterations suggest that these drugs induce changes in protein conformation of the membranes. The membrane binding properties of these drugs may be important in their mechanism of action.     

Hypothetical role of RNA damage avoidance in preventing human disease

Most of nucleic acids damaging agents are not only restricted to DNA but equally affect DNA and RNA molecules. Considering that RNA damage could be very toxic for the cell, a property used by some cancer treatments, it would not be unexpected to find out that several proteins may be involved in RNA damage avoidance mechanisms helping cells to counteract such cytotoxic effects. Up to now, only one specific repair mechanism allowing cells to deal with toxic effects of methylated RNA have been described. However, there are in the literature several data suggesting that this study may only be the tip of the iceberg and that cells might be able to counteract the deleterious effects of a large variety of RNA damage. In this review, we will discuss the different proteins that may be involved in the mechanism of RNA damage avoidance and their potential role in human diseases.     

Use of Tetrahymena pyriformis to Evaluate the Effects of Purine and Pyrimidine Analogs1

SYNOPSIS Eighty-four purine and pyrimidine analogs were evaluated for growth inhibition of Tetrahymena pyriformis. The most toxic were 2-fluoroadenine, 2-fluoroadenosine, 6-methylpurine, a series of 5-fluoropyrimidines, and a series of adenine derivatives substituted in the 9-position. 2-Fluoroadenine was metabolized to the ribonucleoside triphosphate and was incorporated into nucleic acids; its inhibition of growth was reversed by high levels of adenine. 6-Methylthiopurine ribonucleoside was phosphorylated, but only to the monophosphate derivative. Contrasting T. pyriformis with mammalian cells gave clues to the mechanism of action of some of the agents. 6-Mercaptopurine, 6-methylthiopurine ribonucleoside, and 6-thioguanine, all potent pseudofeedback inhibitors of de novo purine biosynthesis in mammalian cells, are not toxic to T. pyriformis, which lacks the de novo purine pathway; this implies that inhibition of de novo purine biosynthesis by them underlies their growth inhibition of mammalian cells.     

Effect of cyclic adenosine-3,5-monophosphate and its dibutyryl analog on macromolecule biosynthesis in actively proliferating cells

The effect of cAMP and its dibutyryl analogue on the biosynthesis of nucleic acids and protein in active proliferating cells was studied. It was shown that cAMP (10(-3)--10(-4)M) caused stimulation of the biosynthesis of DNA and RNA in Ehrlich's ascites carcinoma (EAC) cells and intensification of collagen biosynthesis in the chick embryo cartilage tissue in vitro. Dibutyryl -- cAMP (10(-3)--10(-4)M) has an inhibitory action on the biosynthesis of macromolecules both in EAC cells and embryonic cartilage tissue. Addition of cAMP phosphodiesterase inhibitors together with cAMP to the incubation media prevents the stimulation of macromolecular biosynthesis observed under the influence of cAMP. Studies on cAMP metabolism revealed that this compound is rapidly catabolized to AMP and adenosine. The latter enters the cells and incorporates into the adenyl nucleotide intracellular pool. The stimulant action of exogenous cAMP is related to its extracellular metabolism rather than to the intracellular effects of the nucleotide.     

Evaluation of the cytotoxic mechanisms mediated by the broad-spectrum antitumor alkaloid acronycine and selected semisynthetic derivatives.

Acronycine (I) is a broad-spectrum antitumor agent whose development as a clinically useful agent has been hindered, in part, due to its poor solubility characteristics. With the goal of acquiring information that may prove of value in the development of structurally related compounds of greater clinical utility, mechanistic studies were performed with acronycine (I) and two semisynthetic derivatives, 2-nitroacronycine (II) and acronycine azine (III). These three substances demonstrated cytotoxic activity with several human tumor cell lines (breast, colon, lung, melanoma, KB-3, and drug-resistant KB-V1). Compounds II and III demonstrated greater activity than I, and more detailed studies were performed with cultured human breast cancer cells (UISO-BCA-1). Acronycine azine (III) induced the cells to accumulate in the G0/G1 phase of the cell cycle. It effectively inhibited the in vitro catalytic activities of partially purified DNA and RNA polymerases in a manner that was competitive with respect to DNA substrate. As judged by spectrophotometric titration, compound III interacted with calf thymus DNA, calf liver RNA, and a variety of single- and double-stranded (deoxy)ribonucleotides. Although no nucleic acid base specificity was discernable, this interaction appeared to be related to the cytotoxic mechanism of this dimeric substance. Monomeric compounds I and II did not interact with nucleic acids, but were effective inhibitors of DNA and RNA synthesis as judged by in vitro systems comprised of cultured UISO-BCA-1 cells or homogenates derived from these cells. The relative inhibitory activities of compounds I and II correlated with their cytotoxic activities suggesting a causal relationship. In addition, these two compounds induced cultured cells to accumulate in the phase of the cell cycle wherein the DNA content ranged from 2n-4n (S + G2/M), and inhibited in vitro DNA and RNA synthesis in a manner that was competitive with respect to nucleotide (TTP or UTP) substrate. Compounds I and II demonstrated greater cytotoxic activity with drug-resistant KB-V1 cells as compared with the parent (drug-sensitive) cell line, whereas this was not the case with compound III. Based on these results and previous literature reports, compounds I, II and III are likely to function by multiple mechanisms of action. However, it appears that alteration of nucleic acid metabolism is key to the activity of each of the substances.     

Morphofunctional mitochondrial response to methylglyoxal toxicity in Bufo bufo embryos

Methylglyoxal (2-oxopropanal) is a reactive alpha-oxoaldehyde that can be formed endogenously mainly as a by-product of glycolytic pathway. It is a cytotoxic compound with significant antiproliferative properties as it can bind, under physiological conditions, to nucleic acids and proteins, forming stable adducts. We have recently shown that exogenous methylglyoxal (150-600 microM) is highly toxic for amphibian embryos where it produces, when added to the culture water, inhibition of cell proliferation in the early developmental stages, followed by severe malformations and strongly reduced embryonic viability. In this work we investigate the morphofunctional effect of methylglyoxal on the common toad B. bufo embryo mitochondria in order to verify if its dysmorphogenetic action might be also ascribed to impairment of mitochondrial functions. The mitochondria were isolated from embryos at the developmental stages of morula, neural plate and operculum complete and developing in the presence of 600 microM methylglyoxal. The results show that exogenous methylglyoxal is highly toxic at mitochondrial level, where it produces proliferation, swelling and membrane derangement. As a consequence, mitochondria from treated embryos show decreased oxidative phosphorylation efficiency, as indicated by the significant reduction both of the respiratory control index values and of the embryonic ATP content. On the basis of these data, it is possible that the methylglyoxal-induced embryonic malformations as well as the strongly reduced viability might be also ascribed to energy depletion.     

Neuromelanogenic and Cytotoxic Properties of Canine Brainstem Peroxidase

We have isolated a heme protein from canine midbrains that possesses potent peroxidase activity. This enzyme catalyzes the oxidation of dopamine to neuromelanin in the presence of H2O2. We have further shown that the isolated peroxidase possesses potent cytotoxic activity in the presence of superoxide or H2O2 and Cl-. The enzyme possesses an endogenous NAD(P)H oxidase activity that can promote the cytotoxic activity by virtue of its production of superoxide. Other enzymes such as dihydroorotate dehydrogenase and galactose oxidase, which produce O2- and H2O2, respectively, are also effective in promoting the cytotoxic activity of the brainstem peroxidase. Although rat erythrocytes were routinely used as the target cell, other cell types, including rat hepatoma and mouse neuroblastoma cells, are also susceptible to the toxic action of the peroxidase. The cytotoxic action of the brainstem peroxidase is dramatically enhanced by kainic acid and is significantly enhanced by Mn2+, whereas dopamine was found to be a potent inhibitor of the cytotoxic activity. Based on these findings, we postulate a central role for the brainstem peroxidase in dopamine metabolism as well as in the biochemical and anatomical changes associated with Parkinson's disease.     

Effect of Nitrosative Stress on Extracellular Nucleotide Metabolism in Endothelial Cells

Mechanisms of free radical injury involve chemical modification of proteins, lipid derivatives and nucleic acids and consequent loss of its function. However, specific targets and exact sequence of events has not been fully clarified. We determined whether extracellular enzymes that are involved in adenosine formation such as ecto-5′nucleotidase (e5N) and removal such as extracellular form of adenosine deaminase (eADA) could be affected by peroxynitrite. We used intact cell assay system that involves exposure of cultured HMEC-1 cells to substrates followed by HPLC analysis of conversion of substrates into products. We found that e5N and ADA activities decreased by 20–40% after incubation for 20 or 60 minutes with 30 μM peroxynitrite. Decrease of cellular ATP and NAD was also observed. We conclude that besides other cytotoxic effects modification of extracellular enzymes of nucleotide metabolism could be important target for free radical injury.     

Physiological activity of fascaplisine--an unusual pigment from tropical sea fishes

Various aspects of the physiological activity of fascaplysine, a pigment from tropic sea sponges, were studied. One of the fragments of the chemical structure of the pigment is the indole ring. Ehrlich tumor cells, murine lymphocytes and erythrocytes were used as the biological tests and it was shown that in high doses (up to 50 micrograms/ml) fascaplisine had a low cytotoxic action on the resting cells. When the tumor cells and lymphocytes were subjected to the action of the proliferative and mitogenic stimuli, fascaplisine in doses up to 1 micrograms/ml showed a high inhibitory effect on involvement of labeled thymidine, uridine and leucine into the cell biosynthesis of the macromolecules. No significant antitumor effect of fascaplisine was stated when its in vivo antitumor activity was studied with doses of 5 to 20 mg/kg on a model of Ehrlich ascitic carcinoma. The absence of the antitumor activity is likely to be associated with the observed suppressive action on the immunocompetent cells.     

Fatty acid biosynthesis in Erlich cells. The mechanism of short term control by exogenous free fatty acids.

We have examined the mechanism by which extracellular free fatty acids regulate fatty acid biosynthesis in Ehrlich ascites tumor cells. De novo biosynthesis in intact cells was inhibited by stearate greater than oleate greater than palmitate greater than linoleate. The amount of citrate and long chain acyl-CoA in the cells was not changed appreciably by the addition of free fatty acids to the incubation medium, indicating than free fatty acids do not regulate fatty acid biosynthesis by changing the total intracellular content of these metabolites. By measuring the incorporation of labeled free fatty acids into acyl-CoA, however, it was determined that the fatty acid composition of the acyl-CoA poolwas changed dramatically to reflect the composition of the exogenous free fatty acids. The relative inhibitory effects of different free fatty acids appear to depend on the ability of their acyl-CoA derivatives to regulate acyl-CoA carboxylase activity. The acyl-CoA concentration needed to produce 50% inhibition of purified Ehrlich cell carboxylase was found to be 0.68 mum for stearoyl-CoA, 1.6 mum for oleoyl-CoA, 2.2 mum for palmitoyl-CoA, 23 mum for myristoyl-CoA, 30 mum for lauroyl-CoA, and 37 mum for linoleoyl-CoA. In contrast to their effects on de novo synthesis, all of the free fatty acids added except stearate stimulated chain elongation in intact cells. Microsomal chain elongation, the major system for elongation in Ehrlich cells, also was regulated by the composition of the cellular acyl-CoA pool. Lauroyl-CoA, myristoyl-CoA, and palmitoyl-CoA were good substrates for elongation by isolated microsomes; oleoyl-CoA, and linoleoyl-CoA were intermediate; and stearoyl-CoA was a very poor substrate. We conclude that free fatty acids regulate fatty acid biosynthesis by changing the composition of the cellular acyl-CoA pool. These changes control the rate of malonyl-CoA production and, because of the acyl-CoA substrate specificity of the microsomal elongation system, modulate the amount of malonyl-CoA used for chain elongation.     

二氢硫辛酰转乙酰基酶通过乙酰化磷酸葡糖酸脱氢酶促进核酸合成

丙酮酸脱氢酶复合物(pyruvate dehydrogenase complex,PDC)是位于线粒体内的多酶复合物,催化丙酮酸不可逆地氧化脱羧转为乙酰辅酶A,二氢硫辛酰转乙酰基酶(dihydrolipoyl acetyltransferase,DLAT)是PDC的1个亚基.PDC在细胞线粒体呼吸中发挥关键作用.但是D...     

Chlorpromazine-induced damage on nucleic acids: a combined cytogenetic and biochemical study.

Chlorpromazine is now emerging as an adjuvant chemotherapeutic agent for the treatment of neoplasia. This was further supported in the present study by the following lines of evidence: it was shown that chlorpromazine causes damage in a series of native nucleic acids, though at somewhat high concentrations. Furthermore, chlorpromazine and caffeine were shown to act synergistically to potentiate the cytogenetic effect of adriamycin on human lymphocytes in vitro and on Ehrlich ascites tumour (EAT) cells in vivo. It is suggested that chlorpromazine alone or in combination with caffeine may exert its cytotoxic effect on normal and neoplastic cells not only indirectly, i.e. by facilitating the intracellular retention of adriamycin, but also directly by intercalating into nucleic acids.     

The determination of individual sensitivity to 5-fluorouracil in patients with malignant tumors in different locations]

Possible determination of individual chemosensitivity of tumors to 5-fluorouracil was investigated using new original data and suggestions that lymphocytes of the tumor carrier have an ability to assimilate chemical drugs. Such an ability is likely due to the fact that 5-fluorouracil as an antimetabolite converted its action by inclusion of lymphocytes to the nucleic acid metabolism. The relation between the phenomenon and the chemotherapy efficacy is possibly realized through two mechanisms. One of them is the direct cytotoxic action of chemical drugs on lymphocyte tumor-associated clones resulting in impairment of the tumor growth control. The other is possible participation of lymphocytes in transport of chemical drugs to the tumor tissue. The in vitro model for estimation of tumor carrier chemosensitivity by the level of the nucleic acid metabolism reflects the impact of the immune system on realization of the antitumor effect of chemical drugs.     

Viruses of Entamoeba histolytica IV. Studies on the Nucleic Acids of the Filamentous and Polyhedral Viruses

The nucleic acids of two amoebal viruses were studied by several independent methods. The filamentous virus, V(ABRM), was shown to be inhibited by bromodeoxyuridine, iododeoxyuridine, and cytosine arabinoside. With acridine orange staining, V(ABRM) inclusions appeared greenish-yellow, indicating that these contained double-stranded nucleic acid. The polyhedral virus, V(301), was also inhibited by bromodeoxyuridine, iododeoxyuridine, and cytosine arabinoside. In addition, nucleic acid hybridization showed that a new DNA species was synthesized in infected amoebal cultures. The intracellular localization of this new DNA was consistent with previous electron microscope studies of the cytoplasmic maturation of V(301).