Evaluation of P53 and BAX gene expression and induction of apoptosis and necrosis by the cis-Pt(II) complex of 3-aminoflavone in comparison with cis-diamminedichloroplatinum(II) (cis-DDP) in human lymphocytes

Currently cis-diamminedichloroplatinum(II) (cis-DDP) is one of the most commonly applied compounds in chemotherapy of many types of cancer. However, a drawback is that its effectiveness presents with many side effects. Therefore, human normal lymphocytes were chosen as a model system to study cis-bis(3-aminoflavone)dichloroplatinum(II) (the cis-Pt(II) complex of 3-aminoflavone) in comparison with cis-DDP. We examined the effect of both tested compounds on cell viability and induction of apoptosis and necrosis. Trypan blue and acridine orange/ethidium bromide staining were carried out, as well as quantitative analysis of the apoptotic signal of P53 and BAX induction caused by the cis-Pt(II) complex of 3-aminoflavone in comparison with cis-DDP. cis-DDP induced a decrease of cell viability and led to a higher increase in necrosis and apoptosis than did the cis-Pt(II) complex of 3-aminoflavone. Moreover, at the molecular level cis-DDP increased P53 and BAX expression in comparison with the other tested compound. The cis-Pt(II) complex of 3-aminoflavone showed a weaker genotoxic effect in normal lymphocytes in comparison with cis-DDP, which was a stronger inducer of apoptosis and necrosis.     

Cross-resistance ofEscherichia coli B/r tocis-platinum (II) diamminochloride,UV light and alkylating agents

Gradual transfers of the strain Escherichia coli B/r on M9 agar with increasing concentrations of cis-platinum (II) diamminochloride (cis-Pt(II)) yielded a resistant strain SM 405 capable of growing on liquid M9 medium containing 250 muM cis-Pt(II). The parent strain Escherichia coli B/r is completely inhibited in both division and growth at cis-Pt(II) concentrations as low as 30 muM. The resistant mutant has a longer doubling time than the parent strain. No other differences were found between the two strains. To elucidate the nature of the resistance, the effect of cis-Pt(II) on the survival of the two strains was compared with that of nitrogen mustard, UV light and ethyl methanesulphonate (EMS). The resistant strain SM 405 was found to be more hardened against the lethal action of UV light and nitrogen mustard but less so against EMS. It had also a higher ability of a host-cell reactivation of UV-irradiated phage T3. The different resistance of the B/r and SM 405 strains is probably due to a mutation increasing the effectiveness of the excision repair in the latter.     

Genotoxicity of cis-PtII complex of 3-aminoflavone in comparison with cis-DDP in A549 cells evaluated by comet assay

Cis-diamminedichloroplatinum(II) (cis-DDP) is one of the most successful antineoplastic drugs. However, besides effectiveness it gives many side effects. Therefore, current studies are concentrated on searching for new analogs equally effective in chemotherapy but less toxic. Comparison of genotoxic properties of cis-Pt(II) complex of 3-aminoflavone and cis-DDP in a comet assay with and without H2O2 application was performed in A549 cell line. The higher tail moment values were noticed for the former compound contrary to the latter one in both variants. It suggests mainly DNA breaks (besides cross-links) appearance after cis-Pt(II) complex of 3-aminoflavone application and might indicate DNA degradation in comparison with cis-DDP.     

Effects of antimitotic agents either free or bound to DNA on mouse peritoneal macrophages cultivated in vitro

Mouse peritoneal macrophages were cultivated in vitro and treated with ethidium bromide (EB) or with cis-dichloro-diammine platinum (II) (cis-Pt). EB provokes strong cytological alterations and cell degeneration; cis-Pt was not toxic under our experimental contitions. EB-DNA complex penetrates into the macrophages, is liberated from DNA in vacuoles, then diffuses into the cell and is highly cytotoxic. Cis-Pt-DNA complex also penetrates into the cells, but cis-Pt cannot be released from DNA, cis-Pt-DNA complex accumulates inside cytoplasmic vacuoles but has no cytotoxic activity.     

Induction of apoptosis and necrosis in A549 cells by the cis-Pt(II) complex of 3-aminoflavone in comparison with cis-DDP

Non-small cell lung cancer (NSCLC) includes a group of tumors that respond poorly to drugs. cis-Diamminedichloroplatinum(II) (cis-DDP) toxicity still remains problematic, and not completely solved by the improvement of supportive care. Therefore, the cis-Pt(II) complex of 3-aminoflavone was selected from cis-DDP analogues with a more favourable toxic profile towards normal cells and at least similar or better anti-tumor activity in comparison with cis-DDP. The aim of this research is to compare the ability of the cis-Pt(II) complex of 3-aminoflavone and cis-DDP to induce apoptosis and necrosis in the human non-small cancer cell line A549. Trypan blue dye exclusion, fluorochrome staining (acridine orange/ethidium bromide double staining), MTT and TUNEL (TdT-mediated dUTP Nick-End Labeling) assays were used. The results obtained show that the cis-Pt(II) complex of 3-aminoflavone is more active in inducing apoptosis and necrosis and in decreasing viability in A549 cells than cis-DDP, which suggests that it could be a potential chemotherapeutic drug.     

Properties of 7-substituted deoxyguanosines formed by cis-diamminedichloroplatinum(II)

cis-Diamminedichloroplatinum(II) (cis-Pt) was reacted with deoxyguanosine and guanosine at pH 6 and the reaction products were purified by HPLC. The products were characterized by UV and 1H-NMR spectra and by incubating cis-Pt with 7-methylguanosine, N2-methylguanosine, [8-3H]guanosine and [U-14C]guanosine. The main product was shown to be a cross-link, in which cis-Pt was linked to the N-7 atoms of two guanines. In the other product cis-Pt was bound monofunctionally to the N-7 atom of guanine. The cis-Pt adducts had many unique properties compared to other N-7 alkylated guanosines. When cis-Pt was incubated with [8-3H]guanosine, the products still had their 8-3H-radioactivity. At pH 10 at room temperature the imidazole ring of the monoadduct was not opened in 20 days, while the half-life of imidazole ring-opening for 7-methyldeoxyguanosine was 21.5 min. The half-lives of acid-catalyzed depurination in 0.1 M HCl at 70 degrees C for deoxyguanosine, 7-methyldeoxyguanosine and the monoadduct were 30 s, 48 s and 35 min, respectively.     

Effects of the cis-dichloro-diammino-platinum(II)-deoxyribonucleic acid on normal and cancerous cells]

The effects of cis-dichloro-diammino-platinum(II) (cis-Pt) bound to DNA have been compared to those of free cis-Pt in mouse Ehrlich tumour cells, in peritoneal macrophages and in chick embryo fibroblasts cultivated in vitro. Cis-Pt has no antimitotic activity anymore when linked to DNA. This would be due to the fact that free cis-Pt is not released from cis-Pt-DNA complex inside lysosomes.     

Uvr excision repair protein complex of Escherichia coli binds to the convex side of a cisplatin-induced kink in the DNA.

The Escherichia coli UvrABC endonuclease is capable of initiating the repair of a wide variety of DNA damages. To study the binding of the UvrAB complex to the DNA at the site of a lesion we have constructed a synthetic DNA fragment with a defined cis-diamminedichloroplatinum(II) (cis-Pt).GG adduct. The cis-Pt.GG is the major adduct after treatment of DNA with the antitumor agent cisplatin. Binding to the DNA at the site of the defined lesion was studied with DNase I and MPE.Fe(II) hydroxyl radical footprinting. The results indicate that the UvrAB complex binds to the convex side of the kink in the DNA caused by the cis-Pt.GG adduct. Concerted incisions of the damaged strand by the UvrABC endonuclease were at the 8th phosphodiester bond 5' to and at the 4th bond 3' of the adjacent guanines. An additional incision was found at the 15th phosphodiester bond 5' to the damaged site. This extra incision was stimulated by a high concentration of UvrC.     

The nature of inactivating lesions produced by platinum(II) complexes in phage lambda DNA

Lambda DNA loses transfectivity and acquires interstrand cross-links after treatment with either trans-Pt(II) or cis-Pt(II). With trans-Pt(II) there is close to an equivalence between the fraction of lambda DNA cross-linked and the fraction inactivated. In contrast, with cis-Pt(II) there are approx. 5 inactivating lesions for each lambda DNA interstrand cross-link. These results suggested that trans-PT(II) does not introduce intrastrand inactivating lesions into lambda DNA while cis-Pt(II) does so. To verify this conclusion, the cross-linked and uncross-linked fractions of lambda DNA treated with trans-PT(II) or cis-Pt(II) were separated on alkaline sucrose gradients. After trans-Pt(II) treatment, the uncross-linked fraction of lambda DNA was transfective when renaturated. However after cis-Pt(II) treatment the uncross-linked fraction of lambda DNA was not transfective when renatured. Thiourea treatment restored transfectivity to all inactivated fractions, showing that these lesions are reversible. We conclude that trans-Pt(II) inactivates lambda DNA primarily by introducing interstrand cross-links but that cis-Pt(II), although it also introduces interstrand cross-links, inactivates lambda DNA primarily by introducing intrastrand lesions.     

Mono- and bifunctional binding of cis-diamminedichloroplatinum(II) to dinucleotides

cis-Diamminedichloroplatinum(II) (cis-Pt) was reacted with four homodinucleotides (GpG, ApA, CpC, and UpU) and six heterodinucleotides (GpC, CpG, GpU, UpG, GpA, and ApG) at pH 6, and the reaction products were purified by HPLC. The most important products were characterized by 1H-NMR spectra. In all the heterodinucleotides except the ones containing uridine the main Pt-adduct was an intramolecular cross-link, but monofunctional adducts and intermolecular cross-links were also detected. Intramolecular cross-links were also formed with GpU and UpG but the amounts of them were about the same as the amounts of intermolecular cross-links. In the case of homodinucleotides GpG gave almost entirely intramolecular cross-links, in which cis-Pt was chelated between the N-7 atoms of two guanines. cis-Pt reacted also with ApA forming both monofunctional and bifunctional Pt-adducts. The main adducts were intramolecular cross-links. cis-Pt reacted equally well with all guanosine-containing dinucleotides, while the reaction with ApA was much slower. With CpC and UpU no reaction products were formed.     

A theoretical model for the binding of cis-Pt(NH3)2(+2) to DNA

The binding of cis-Pt(NH3)2B1B2 to the bases B1 and B2, i.e., guanine (G), cytosine (C), adenine (A), and thymine (T), of DNA is studied theoretically. The components of the binding are analyzed and a model structure is proposed for the intrastrand binding to the dB1pdB2 sequence of a kinked double helical DNA. Quantum mechanical calculations of the ligand binding energy indicates that cis-Pt(NH3)2(+2) (cis-PDA) binds to N7(G), N3(C), O2(C), O6(G), N3(A), N7(A), O4(T) and O2(T) in order of decreasing binding energy. Conformational analysis provides structures of kinked DNA in which adjacent bases chelate to cis-PDA. Only bending toward the major groove allows the construction of acceptable square planar complexes. Examples are presented for kinks of -70 degrees and -40 degrees at the receptor site to orient the base pairs for ligand binding to B1 and B2 to form a nearly square planar complex. The energies for complex formation of cis-PDA to the various intra-strand base sites in double stranded DNA are estimated. At least 32 kcal/mole separates the energetically favorable dGpdG.cis-PDA chelate from the dCpdG.cis-PDA chelate. All other possible chelate structures are much higher in energy which correlates with their lack of observation in competition with the preferred dGpdG chelate. The second most favorable ligand energy occurs with N3(C). A novel binding site involving dC(N3)pdG(N7) is examined. Denaturation can result in an anti----syn rotation of C about its glycosidic bond to place N3(C) in the major groove for intrastrand binding in duplex DNA. This novel intrastrand dCpdG complex and the most favored dGpdG structure are illustrated with stereographic projections.     

Reactions of the UVRABC excision nuclease with DNA damaged by diamminedichloroplatinum(II).

Mutants of Escherichia coli, which are blocked in excision repair (uvrA6, uvrB5, or uvrC34) are exceptionally sensitive to the antitumor drug cis-Pt(II)(NH3)2Cl2 (cis-DDP) but not the trans isomer. Plasmid DNA, damaged by either the cis or trans compound and treated with the UVRABC excision nuclease was cut as shown by conversion of supercoiled DNA to relaxed forms. All three protein products of the uvrA, uvrB, and uvrC genes were required for incision. End-labeled fragments damaged with cis-DDP and reacted with the UVRABC nuclease were cut at the 8th phosphodiester bond 5' and at the 4th phosphodiester bond 3' to adjacent GG's. DNA treated with trans-DDP was not cut appreciably at adjacent GG's by the repair enzyme as subsequent analysis of reaction products after enzyme digestion gave a pattern similar to those obtained with control untreated fragments. The results indicate that the UVRABC nuclease may promote cell survival by the removal of adjacent GG's which are crosslinked by cis-Pt(II)(NH3)2Cl2.     

Hydrolytic cleavage of DNA by quercetin zinc(II) complex

Quercetin zinc(II) complex was investigated focusing on its hydrolytic activity toward DNA. The complex successfully promotes the cleavage of plasmid DNA, producing single and double DNA strand breaks. The amount of conversion of supercoiled form (SC) of plasmid to the nicked circular form (NC) depends on the concentration of the complex as well as the duration of incubation of the complex with DNA. The rate of conversion of SC to NC is 1.68x10(-4) s(-1) at pH 7.2 in the presence of 100 microM of the complex. The hydrolytic cleavage of DNA by the complex is supported by the evidence from free radical quenching, thiobarbituric acid-reactive substances (TBARS) assay, and T4 ligase ligation.     

Synthesis and characterization of a d(ApG) platinated nonanucleotide duplex.

The nonamer 5'd(CTCAGCCTC) 3' 1 has been reacted with cis-diamminediaquaplatinum(II) in water at pH 4.2. The major reaction product was shown by enzymatic digestion and 1H NMR to be the d(ApG)cis-Pt(NH3)2 chelate [cis-Pt(NH3)2[d(CTCAGCCTC)-N7(4),N7(5)]] 1-Pt. When mixed with its complementary strand 2, 1-Pt forms a B DNA type duplex 3-Pt with a Tm of 35 degrees C (versus 58 degrees C for the unplatinated duplex). The NMR study of the exchangeable protons of 3-Pt revealed that the helix distortion is localized on the CA*G*-CTG moiety (the asterisks indicating the platinum chelation sites) with a strong perturbation of the A*(4)T(15) base pair related to a large tilt of A*(4).     

Differential sensitivity of Fanconi anaemia lymphocytes to the clastogenic action of cis-diamminedichloroplatinum (II) and trans-diamminedichloroplatinum (II)

Summary Fanconi anaemia (FA) lymphocytes were tested for their susceptibility to chromosomal breakage by cis-diamminedichloroplatinum (II) [cis-Pt(II)] and its stereoisomer trans-diamminedichloroplatinum (II) [trans-Pt(II)]. Unlike trans-Pt(II), which is a rather inefficient clastogen, cis-Pt(II) is very efficient in inducing chromosomal breakage in FA cells at concentrations that hardly affect control cells. As both cis-Pt(II) and trans-Pt(II) are capable of inducing DNA interstrand crosslinks but only cis-Pt(II) can induce DNA intra-strand crosslinks, this result suggests that FA cells may be specifically sensitive to the intrastrand type of DNA crosslink.     

Transduction of plasmid antibiotic resistance determinants with pseudo-T-even bacteriophages

Transduction of antibiotic resistance determinants of the plasmid pBR322 with pseudoT-even bacteriophages RB42, RB43, and RB49 was studied. It is established that antibiotic resistance determinants of plasmid pBR322 from Escherichia coli recA(+)- and recA(-)-donor strains do not differ significantly in respect to the efficiency of transduction. Amber mutants RB43-21, RB43-33, and a double amber mutant RB43am21am33 were obtained. These mutants facilitated transduction experiments in some cases. Transduction of antibiotic resistance markers of the vector plasmid pBR325 and recombinant plasmid pVT123, containing a DNA fragment with hoc segE uvsW genes of phage T4, was studied. The frequency of appearance of transductants resistant to pseudoT-even bacteriophages used in transduction was determined, and the sensitivity of resistant transductants to 32 RB bacteriophages and also to phages lambda, T2, T4, T5, T6, T7, and BF23 was estimated. The efficiency of plating pseudoT-even bacteriophages RB42 and RB43 on strain E. coli 802 himA hip carrying mutations in genes that encode subunits of the Integration Host Factor (IHF) was shown to be higher than on isogenic strain E. coli 802. The growth of pseudoT-even bacteriophages limited in vivo by modification-restriction systems of chromosomal (EcoKI, EcoBI), phage (EcoP1I), and plasmid (EcoRI, EcoR124I, and EcoR124II) localization was analyzed. It was shown that these phages were only slightly restricted by the type I modification-restriction systems EcoBI, EcoR124I, and EcoR124II. Phage RB42 was restricted by systems EcoKI, EcoP1I, and EcoRI; phage RB43, by systems EcoKI and EcoRI; and phage RB49, by the EcoRI modification-restriction system.     

T4 Phage and Its Head Surface Proteins Do Not Stimulate Inflammatory Mediator Production

Viruses are potent activators of the signal pathways leading to increased cytokine or ROS production. The effects exerted on the immune system are usually mediated by viral proteins. Complementary to the progress in phage therapy practice, advancement of knowledge about the influence of bacteriophages on mammalian immunity is necessary. Particularly, the potential ability of phage proteins to act like other viral stimulators of the immune system may have strong practical implications for the safety and efficacy of bacteriophage therapy. Here we present studies on the effect of T4 phage and its head proteins on production of inflammatory mediators and inflammation-related factors: IL-1α, IL-1β, IL-2, IL-6, IL-10, IL-12 p40/p70, IFN-γ, TNF-α, MCP-1, MIG, RANTES, GCSF, GM-CSF and reactive oxygen species (ROS). Plasma cytokine profiles in an in vivo mouse model and in human blood cells treated with gp23*, gp24*, Hoc and Soc were evaluated by cytokine antibody arrays. Cytokine production and expression of CD40, CD80, CD86 and MHC class II molecules were also investigated in mouse bone marrow-derived dendritic cells treated with whole T4 phage particle or the same capsid proteins. The influence of T4 and gp23*, gp24*, Hoc and Soc on reactive oxygen species generation was examined in blood cells using luminol-dependent chemiluminescence assay. In all performed assays, the T4 bacteriophage and its capsid proteins gp23*, gp24*, Hoc and Soc did not affect production of inflammatory-related cytokines or ROS. These observations are of importance for any medical or veterinary application of bacteriophages.     

Differential response to mitomycin-C- and cis-diamminedichloroplatinum(II)-induced damage in normal human fibroblasts during confluent holding

Confluent cultures of normal human fibroblasts were treated with the chemotherapeutic agents, mitomycin C (MMC) and cis-diamminedichloroplatinum(II) (cis-Pt(II]. This treatment induced a decrease in clonogenic cell survival. Recovery of the cytotoxic effect was observed in the case of cis-Pt(II)-treated cultures maintained at confluence for 1-5 days. No such recovery was observed after treatment with MMC. These data suggest that contrary to potential lethal damage induced by cis-Pt(II) which is repaired in confluent cells, DNA damage induced by MMC is not repaired in confluent cells.     

Interactions of competent Streptococcus sanguis (Wicky) cells with native or denatured, homologous or heterologous deoxyribonucleic acids.

Competent cell-deoxyribonucleic acid (DNA) interactions were examined using tritium-labeled homologous or heterologous native or denatured DNAs and competent Streptococcus sanguis Wicky cells (strain WE4). The DNAs used were extracted from WE4 cells, Escherichia coli B cells, and E. coli bacteriophages T2, T4, T6, and T7. The reactions examined were: (i) total DNA binding, (ii) deoxyribonuclease-resistant DNA binding, and (iii) the production of acid-soluble products from the DNA. Optimal temperatures for the reactions were as follows: reaction (i), between 30 and 40 degrees C; reaction (ii), 30 degrees C; and reaction (iii), greater than 40 degrees C. The rates for the reactions (expressed as molecules of DNA that reacted per minute per colony-forming unit) did not vary greatly from one DNA source to another. With a constant competent cell concentration and differing DNA concentrations below a saturation level (from a given source), a different but constant fraction of the added DNA was cell bound, deoxyribonuclease resistant, and degraded to acid-soluble products. In experiments where the number of competent cells was varied and the DNA concentration was held constant, again essentially the same result was obtained. The extent of reactions (i), (ii), and (iii) depended upon the numbers as well as the source of DNA molecules applied to competent cells. Calcium ion essential for native DNA-cell reactions was also found essential for denatured DNA-cell reactions. Data obtained from competition experiments lead to the conclusion that competent WE4 cells contain specific sites for native as well as denatured DNAs.     

Comparison of the physical properties and assembly pathways of the related bacteriophages T7, T3 and phi II

To understand constraints on the evolution of bacteriophage assembly, the structures, electrophoretic mobilities (mu) and assembly pathways of the related double-stranded DNA bacteriophages T7, T3 and phi II, have been compared. The characteristics of the following T7, T3 and phi II capsids in these assembly pathways have also been compared: (1) a DNA-free procapsid (capsid I) that packages DNA during assembly; (b) a DNA packaging-associated conversion product of capsid I (capsid II). The molecular weights of the T3 and phi II genomes were 25.2 X 10(6) and 25.9 (+/- 0.2) X 10(6) (26.44 X 10(6) for T7, as previously determined), as determined by agarose gel electrophoresis of intact genomes. The radii of T7, T3 and phi II bacteriophages were indistinguishable by sieving during agarose gel electrophoresis (+/- 4%) and measurement of the bacteriophage hydration (+/- 2%) (30.1 nm for T7, as previously determined). Assuming a T = 7 icosahedral lattice for the arrangement of the major capsid subunits (p10A) of T7, T3 and phi II best explains these data and data previously obtained for T7. At pH 7.4 and an ionic strength of 1.2, the solid-support-free mu values (mu 0 values) of T7, T3 and phi II bacteriophages, obtained by extrapolation of mu during agarose gel electrophoresis to an agarose concentration of 0 and correction for electro-osmosis, were -0.71, -0.91 and -1.17(X 10(-4) cm2V-1 s-1. The mu 0 values of T7, T3 and phi II capsids I were -1.51, -1.58 and -2.07(X 10(-4] cm2V-1 s-1. For the capsids II, these mu 0 values were -0.82, -1.07 and -1.37(X 10(-4] cm2V-1 s-1. The tails of all three bacteriophages were positively charged and the capsid envelopes (heads) were negatively charged. In all cases the procapsid had a negative mu 0 value larger in magnitude than the negative mu 0 value for bacteriophage or capsid II. A trypsin-sensitive region in capsid I-associated, but not capsid II-associated, T3 p10A was observed (previously observed for T7). The largest fragment of trypsinized capsid I-associated p10A had the same molecular weight in T7 and T3, although the T3 p10A is 18% more massive than the T7 p10A. It is suggested that the trypsin-resistant region of capsid I-associated p10A determines the radius of the bacteriophage capsid.