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A capillary zone electrophoresis-laser-induced fluorescence detection (CZE-LIF) method was developed for the simultaneous analysis of disaccharides derived from heparan sulfate, chondroitin sulfate/dermatan sulfate, hyaluronan, and keratan sulfate. Glycosaminoglycans (GAGs) were first depolymerized with the mixture of GAG lyases (heparinase I, II, III and chondroitinase ABC and chondroitinase AC II) and GAG endohydrolase (keratinase II) and the resulting disaccharides were derivatized by reductive amination with 2-aminoacridone. Nineteen fluorescently labeled disaccharides were separated using 50 mM phosphate buffer (pH 3.3) under reversed polarity at 25 kV. Using these conditions, all the disaccharides examined were baseline separated in less then 25 min. This CZE-LIF method gave good reproducibility for both migration time (a parts per thousand currency sign1.03 % for intraday and a parts per thousand currency sign4.4 % for interday) and the peak area values (a parts per thousand currency sign5.6 % for intra- and a parts per thousand currency sign8.69 % for interday). This CZE-LIF method was used for profiling and quantification of GAG derivative disaccharides in bovine cornea. The results show that the current CZE-LIF method offers fast, simple, sensitive, reproducible determination of disaccharides derived from total GAGs in a single run.

From previous thermal and photoinduced charge-transfer reactions in duplex DNA there is accumulative evidence for an attenuation parameter beta of the distance dependence in the range 0.6-0.8 A(-1), with the exception of one specific system exhibiting beta = 1.5 A(-1) which is reinvestigated in this paper. Femtosecond to nanosecond time-resolved pump-probe spectroscopy has been used to follow photoinduced charge-shift dynamics in DNA duplexes containing a covalently appended, protonated 9-alkylamino-6-chloro-2-methoxyacridine chromophore. This acridine derivative (X+) resides in the DNA duplex at a specific abasic site, which is highly defined as reflected in the monoexponentiality of the kinetics. In the presence of only neighboring A:T base pairs, no charge transfer occurs within the excited-state lifetime (18 ns) of the chromophore. However, the presence of a guanine nucleobase as either a nearest neighbor or with one interspersed A:T base pair does result in fluorescence quenching. In the case of nearest neighbors, the intermediate radical state X* is formed within 4 ps and decays on the 30 ps time scale. Placing one A:T base pair between the X+ and guanine slows down the forward transfer rate by 3 orders of magnitude, corresponding to an apparent beta value of2.0 A(-1). This dramatic decrease in the rate is due to a change in charge-transfer mechanism from a (nearly) activationless to a thermally activated regime in which the forward transfer is slower than the back transfer and the X* state is no longer observed. These observations indicate that the distance dependence of charge injection in the X+-labeled DNA duplex is not solely caused by a decrease in electronic couplings but also by a concomitant increase of the activation energy with increasing distance. This increase in activation energy may result from the loss of driving force due to excited-state relaxation competing with charge transfer, or reflect distance-dependent changes in the energetics, predominantly of the low-frequency reorganization energy in this charge-shift reaction, on purely electrostatic grounds. To test the hypothesis of distance-dependent activation energy, guanine has been replaced by 7-deazaguanine, its easier-to-oxidize purine analogue. In these duplexes, a similar change of charge-transfer mechanism is found. However, consistent with an a priori larger driving force this change occurs at a larger donor-acceptor separation than in the X+-guanine systems. Independent of the detailed contributions to the distance-dependent activation energy, this phenomenon illustrates the complex nature of experimental beta values.

We report a study on the absorptive and emissive properties of 9-acridinones, 9-thioacridinones and 9-aminoacridines including six crown ether derivatives. The effect of solvents and of the addition of cations (Na+, K+, Ca2+ and Mg2+) on these properties has been studied. The absorption of the crown ether derivative of 9-thioacridinone is sensitive to solvents while the fluorescence of crown ethers derived from 9-aminoacridines shows some specificity towards cations. Empirical modeling was used to discuss the emission characteristics of these compounds.

In the fight against malaria, chemotherapy using bisacridines may represent an alternative method to overcoming chloroquine-resistance. Eight bis(9-amino-6-chloro-2-methoxyacri-dines), in which acridine moieties were linked by polyamines substituted with a side chain, were tested for their in-vivo activity upon mice infected by Plasmodium berghei. Three of the compounds revealed antimalarial activity but no relationship could be deduced from a comparison of in-vitro and in-vivo activities. N-alkylation of the central amino group generated toxicity and, therefore, only compounds N-acylated in this position can be selected as leads.

Purpose: To determine the ability of WMC26, a prototypic bisimidazoacridone (BIA), to induce apoptosis in sensitive colon adenocarcinoma cells and to advance the hypothesis that cancer cells that are growth-arrested by WMC26 are predisposed to undergo apoptotic death by abrogators of cell cycle checkpoints. Methods: The antiproliferative activity of WMC26 was examined in detail by a 4-day MTT assay, cell counting, BrdU incorporation and a two-color LIVE/DEAD assay. To detect apoptosis a number of established techniques were used, including gel electrophoresis, flow cytometry, and confocal laser microscopy of treated cells. The activity of senescence-associated β-galactosidase in treated cells was also analyzed. Results: WMC26, at physiological concentrations, induced complete and longlasting growth arrest of HCT116 cells in culture but did not trigger cell death. The growth-arrested cells (blocked at G1 and G2/M cell cycle checkpoints) did not synthesize DNA but were metabolically active and had intact plasma membranes. Although they resembled the senescence-like phenotype reported to be induced by treatment with some antitumor agents, the cells did not express senescence-associated β-galactosidase, an indicator of the senescence-like state. Treatment of WMC26 growth-arrested cells with 1 µM UCN-01, an abrogator of the G2/M checkpoint, caused a very rapid (1 h) change in morphology and cell death within 72 h. Conclusions: BIAs do not induce apoptosis in sensitive colon tumor cells. They are highly cytostatic but only marginally toxic to the cells even at concentrations 100-fold higher than those sufficient for complete growth arrest. In this respect WMC26 differs from some other DNA-interacting antitumor agents that produce cell growth arrest at low concentrations but are toxic at higher doses. The complete growth arrest induced by WMC26 in colon cancer cells sensitized them to apoptotic death induced by UCN-01. This finding suggests that a combination of WMC26 and cyclin-dependent kinase inhibitors may be an attractive treatment method for colon cancer that utilizes the highly tumor-selective activity of WMC26.

The imidazoacridinone derivative, C-1311, is a new antitumour agent that exhibits strong antitumour activity against experimental colorectal cancer and has been selected for entry into clinical trial. The compound has previously been shown to have DNA non-covalent binding properties in vitro and to bind irreversibly to DNA of tumour cells. The latter effect has also been observed in a cell-free system, but only in the presence of activated enzymes. The present studies were aimed at finding out whether and in what way the enzymatic activation of C-1311 and its non-covalent binding to DNA influence or depend on each other. Enzymatic activation was performed with a model system containing horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) and was followed by UV-VIS spectroscopy and by HPLC with UV-VIS and electrospray ionisation mass spectrometry detection. DNA non-covalent binding was studied in the cell-free system by means of an unwinding assay and UV-VIS spectroscopy. It was shown that C-1311 was oxidised by the HRP/H2O2 system in a manner dependent on the drug:H2O2 ratio. In the case of ratios of 1:3 and 1:5, the reaction gave highly reactive species that were quickly transformed into the further products p2 and p3 that were unable to intercalate into DNA. In the presence of DNA, C-1311 first intercalated into DNA and the intercalated compound was then oxidised. This oxidation was directed to only one product. Therefore, DNA seems to play the role of a "scavenger" of the reactive oxidation product(s) yielded from the intercalated drug and prevents its further deactivation. We conclude that, under the conditions studied, intercalation of C-1311 into DNA is followed by its HRP-mediated activation, giving rise to the intercalated species that might irreversibly bind to DNA. Since peroxidase-type enzymes are present in the cell nucleus, the proposed sequence of events may also be expected to take place in the cellular environment in vivo.

A 14 nt DNA sequence 5′-AGAATGTGGCAAAG-3′ from the zinc finger repeat of the human KRAB zinc finger protein gene ZNF91 bearing the intercalator 2-methoxy,6-chloro,9-amino acridine (Acr) attached to the sugar–phosphate backbone in various positions has been shown to form a specific triple helix (triplex) with a 16 bp hairpin (intramolecular) or a two-stranded (intermolecular) duplex having the identical sequence in the same (parallel) orientation. Intramolecular targets with the identical sequence in the antiparallel orientation and a non-specific target sequence were tested as controls. Apparent binding constants for formation of the triplex were determined by quantitating electrophoretic band shifts. Binding of the single-stranded oligonucleotide probe sequence to the target led to an increase in the fluorescence anisotropy of acridine. The parallel orientation of the two identical sequence segments was confirmed by measurement of fluorescence resonance energy transfer between the acridine on the 5′-end of the probe strand as donor and BODIPY-Texas Red on the 3′-amino group of either strand of the target duplex as acceptor. There was full protection from OsO4-bipyridine modification of thymines in the probe strand of the triplex, in accordance with the presumed triplex formation, which excluded displacement of the homologous duplex strand by the probe–intercalator conjugate. The implications of these results for the existence of protein-independent parallel triplexes are discussed.

We highlight selected contributions of Dr. Richard Setlow that contributed to our earlier understanding of excision repair processes and set the stage for dissecting nucleotide excision repair (NER) in mammalian cells through molecular genetics. More than 20 years ago, large-scale screens for UV-sensitive mutants of hamster CHO cells isolated approximately 200 mutants, many of which were assigned to the XPD/ERCC2 complementation group, but the nature of the mutations was not determined. The XPD protein performs not only an essential viability function as a structural component of transcription initiation factor TFIIH, but also an NER function as a 5' to 3' DNA helicase within TFIIH that unwinds DNA on the 3' side of bulky lesions. Alterations in these XPD functions are responsible for three UV-sensitivity genetic disorders that have distinguishable clinical features. In this study, we sequenced six UV-sensitive ICR170-induced Chinese hamster ovary (CHO) cell mutants that previously were assigned to the XPD complementation group to determine whether they carry frameshift mutations. All six mutants show 3- to 5-fold increased hypersensitivity to UV irradiation, similar to the XPD mutant prototype UV5. Even though ICR170 is a strong frameshift mutagen, all six cell lines contain base substitution mutations, five of which are unique among all mutations identified so far in human and rodent cells. The sixth mutation was identical to the R75W mutation previously found in CHO UVL-1. The results presented here contribute to a mutation database that should prove useful in structure-function studies of this unique DNA-structure-specific helicase and its complex mutant phenotypes.

The structures of the complexes formed between 9-amino-[N:-(2-dimethyl-amino)butyl]acridine-4-carboxamide and d(CG(5Br)UACG)(2) and d(CGTACG)(2) have been solved by X-ray crystallography using MAD phasing methodology and refined to a resolution of 1.6 A. The complexes crystallised in space group C222. An asymmetric unit in the brominated complex comprises two strands of DNA, one disordered drug molecule, two cobalt (II) ions and 19 water molecules (31 in the native complex). Asymmetric units in the native complex also contain a sodium ion. The structures exhibit novel features not previously observed in crystals of DNA/drug complexes. The DNA helices stack in continuous columns with their central 4 bp adopting a B-like motif. However, despite being a palindromic sequence, the terminal GC base pairs engage in quite different interactions. At one end of the duplex there is a CpG dinucleotide overlap modified by ligand intercalation and terminal cytosine exchange between symmetry-related duplexes. A novel intercalation complex is formed involving four DNA duplexes, four ligand molecules and two pairs of base tetrads. The other end of the DNA is frayed with the terminal guanine lying in the minor groove of the next duplex in the column. The structure is stabilised by guanine N7/cobalt (II) coordination. We discuss our findings with respect to the effects of packing forces on DNA crystal structure, and the potential effects of intercalating agents on biochemical processes involving DNA quadruplexes and strand exchanges. NDB accession numbers: DD0032 (brominated) and DD0033 (native).

The effects of environmental estrogenic compounds, soy isoflavone mixture (SI), genistein (GEN), and nonylphenol (NP), and the possible goitrogen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), on thyroid carcinogenesis were investigated in ovariectomized (OVX) female rats. Five-week-old OVX F344 rats were given a single subcutaneous injection of N-bis(2-hydroxypropyl)nitrosamine (DHPN; 2400 mg / kg, body weight) or vehicle alone. Starting 1 week later, GEN (250 or 25 ppm in diet), SI (400 ppm in diet), NP (250 or 25 ppm in diet), MX (30 ppm, in drinking water), sulfadimethoxine (SDM), a known thyroid tumor-promoter (1000 ppm in drinking water), or beta-estradiol 3-benzoate (EB), a synthetic estrogen (0.5 mg in cholesterol pellet, s.c.) were administered for 12 weeks. SDM and EB were included as positive controls. At sacrifice the major organs including the thyroid, pituitary, liver, kidney, uterus, vagina, brain and pancreas were collected and histopathological observation was performed. Thyroid weights were significantly increased (P < 0. 001) only in the SDM treatment group and pituitary weights were elevated with SDM (P < 0.05) and EB (P < 0.001). Kidney and uterus weights were also significantly increased (P < 0.05) by EB. Histopathologically, proliferative lesions of the thyroid were only observed in the SDM treatment group and of the pituitary in the SDM or EB treatment groups. Renal tubule lesions, uterine squamous metaplasia, vaginal keratinization and telangiectasia of pancreatic islets were also observed with EB. There were no organ weight changes or histopathological lesions in the major organs, including the thyroid, in the GEN, SI, MX or NP treatment groups. Our results thus indicated a lack of modifying effects on thyroid carcinogenesis in female OVX rats, in agreement with our previous finding in males.

The antigenic determinants of mAbs against subunit c of the Escherichia coli ATP synthase were mapped by ELISA using overlapping synthetic heptapeptides. All epitopes recognized are located in the hydrophilic loop region and are as follows: 31-LGGKFLE-37, 35-FLEGAAR-41, 36-LEGAAR-41 and 36-LEGAARQ-42. Binding studies with membrane vesicles of different orientation revealed that all mAbs bind to everted membrane vesicles independent of the presence or absence of the F1 part. Although the hydrophilic region of subunit c and particularly the highly conserved residues A40, R41, Q42 and P43 are known to interact with subunits gamma and epsilon of the F1 part, the mAb molecules have no effect on the function of F0. Furthermore, it could be demonstrated that the F1 part and the mAb molecule(s) are bound simultaneously to the F0 complex suggesting that not all c subunits are involved in F1 interaction. From the results obtained, it can be concluded that this interaction is fixed, which means that subunits gamma and epsilon do not switch between the c subunits during catalysis and furthermore, a complete rotation of the subunit c oligomer modified with mAb(s) along the stator of the F1F0 complex, proposed to be composed of at least subunits b and delta, seems to be unlikely.

C1311 is a novel therapeutic agent with potent activity against experimental colorectal cancer that has been selected for entry into clinical trial. The compound has previously been shown to have DNA-binding properties and to inhibit the catalytic activity of topoisomerase II. In this study, cellular uptake and mechanisms by which C1311 interacts with DNA and exerts cytotoxic effects in intact colon carcinoma cells were investigated. The HT29 colon cancer cell line was chosen to follow cellular distribution of C1311 over a time course of 24 h at drug concentrations that just inhibited cell proliferation by 50% or 100%. Nuclear uptake of C1311 and co-localization with lysosomal or mitochondrial dyes was examined by fluorescence microscopy and effects on these cellular compartments were determined by measurement of acid phosphatase levels, rhodamine 123 release or DNA-binding behaviour. The strength and mode of DNA binding was established by thermal melting stabilization, direct titration and viscometric studies of host duplex length. The onset of apoptosis was followed using a TUNEL assay and DNA-fragmentation to determine a causal relationship of cell death. Growth inhibition of HT29 cells by C1311 was concomitant with rapid drug accumulation in nuclei and in this context we showed that the compound binds to duplex DNA by intercalation, with likely A/T sequence-preferential binding. Drug uptake was also seen in lysosomes, leading to lysosomal rupture and a marked increase of acid phosphatase activity 8 h after exposure to C1311 concentrations that effect total growth inhibition. Moreover, at these concentrations lysosomal swelling and breakdown preceded apoptosis, which was not evident up to 24 h after exposure to drug. Thus, the lysosomotropic effect of C1311 appears to be a novel feature of this anticancer agent. As it is unlikely that C1311-induced DNA damage alone would be sufficient for cytotoxic activity, lysosomal rupture may be a critical component for therapeutic efficacy. © 1999 Cancer Research Campaign

An ultrasensitive capillary electrophoretic method for separating the variously sulfated chondroitin/dermatan sulfate-derived delta-disaccharides after digestion with chondro/dermatolyases and derivatization with the fluorophore 2-aminoacridone is described. All known mono-, di- and tri-sulfated delta-disaccharides were completely separated using 15 mM orthophosphate buffer (pH 3.0) at 20 kV without any interference of the excess derivatizing reagent. They were detected at the anode (reversed polarity) using either an Ar-ion laser-induced fluorescence (LIF) detector (excitation wavelength 488 nm) or a UV detector. The sensitivity obtained by LIF (0.51 pmol/l) was at least 100 and 10 times higher as compared to those obtained by UV detection at 232 nm of underivatized delta-disaccharides and at 254 nm of those derivatized with aminoacridone, respectively. The method has been easily applied to the analysis of chondroitin/dermatan sulfates from various tissues at the attogram level, including chondrotin/dermatan sulfates from normal and aneurysmal human abdominal aortas.

The acetylcholinesterase reversible inhibitor N -octyl-1,2,3,4-tetrahydro-9-aminoacridine (THA-C8) is a new synthesized derivative of tacrine (THA) characterized by an alkyl chain in the molecular structure which ameliorates the penetrability of the compound into the central nervous system. THA-C8 (0.1–5 mg/kg) significantly reduced spontaneous locomotor activity in CD1 mice at a dose of 3 mg/kg. Moreover, THA-C8 (0.2–2 mg/kg) significantly improved shuttle-box avoidance acquisition at doses (0.25, 0.3, 1 mg/kg) not affecting locomotion and that are much lower than the doses reported to be effective for THA in animal models. From the data reported it seems that the new compound could be interesting for therapeutic purposes.

Oligosaccharides released from porcine thyroglobulin were first derivatised with 2-aminoacridone (2-AMAC) and analysed by capillary electrophoresis to determine the complexity of this glycan pool. The same glycan mixture was then subjected to either a sialidase digest or a sialidase and fucosidase digest prior to derivatisation with 2-AMAC and analysis by normal phase high pressure liquid chromatography (HPLC). Comparison of the three chromatographic profiles with known standards allowed an initial identification of the glycan structures. The 2-AMAC derivatised glycans were then collected from HPLC for matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) analysis and the molecular weights of predicted structures were confirmed. This study demonstrates that a two enzyme array and subsequent MALDI-TOF analysis can be used successfully to assign the major glycans present in a complex mixture.

The presented study concentrated on the oxidative enzymatic transformation of six imidazoacridinone derivatives exhibiting different antitumour activity. Horseradish peroxidase was applied as an enzymatic model system. The investigations aimed to evaluate: (1) whether the studied compounds can undergo oxidative biotransformation; (2) whether the susceptibility to such biotransformation relates to the structure and antitumour activity of these compounds; and (3) which elements of imidazoacridinone structure are involved in this kind of transformation. The reaction courses were followed by three methods: UV-VIS spectroscopy, electron paramagnetic resonance and high-performance liquid chromatography. It was shown that all the imidazoacridinones studied underwent enzymatic oxidation resulting in the formation of several products, spectra of which revealed that imidazoacridinone chromophore as well as alkylamino side-chain were involved in these biotransformations. The susceptibility to enzymatic oxidation turned out to be well correlated with antitumour activity of these compounds. It was demonstrated that the highly active antitumour 8-hydroxy derivatives underwent oxidative transformation far more readily than the less active 8-methoxy derivatives and analogues without substituent in position 8. The results indicated that the oxidation pathway of 8-hydroxy compounds was different from those observed for the remaining imidazoacridinones studied. It also differed from the pathway proposed earlier for mitoxantrone. Moreover, it was find out that not only the rate but also the mechanism of horseradish oxidation of 8-hydroxy derivatives depended on the reaction conditions. In the presence of excess of hydrogen peroxide, the drugs were exceptionally reactive giving rise to the mixture of many unstable products, among which compounds with both changed and unchanged chromophore structure were formed. However, the equimolar ratio of drug and hydrogen peroxide led to stable products, which resulted from the oxidation in aminoalkyl side-chain. The possible structures of products of imidazoacridinone enzymatic oxidation are discussed. In conclusion, the results presented in this paper indicate that the oxidative metabolic activation of imidazoacridinones may represent the crucial step in their biological action.

Oligosaccharides released from human transferrin have been derivatized with 2-aminoacridone (2-AMAC) prior to analysis by either reversed- or normal-phase high-performance liquid chromatography. These separation methods are complementary and allow distinction between isomeric mono- and disialylated oligosaccharides, which terminate with Sia alpha 2-6Gal or Sia alpha 2-3Gal at the nonreducing end. Collected fractions of 2-AMAC-derivatized glycans were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, before and after desialylation.

Administration of sublethal doses of endotoxin produces tolerance to subsequent oxidative stress in diverse animal models. Although endotoxin induces antioxidant enzymes, particularly manganous superoxide dismutase (Mn-SOD), the phenomenon of tolerance remains incompletely understood. Previously I determined that endotoxin treatment in rats increased lung mitochondrial respiration-dependent (i.e., independent of Mn-SOD) scavenging of superoxide anion. Because nonenzymatic scavenging of superoxide anion correlates with the mitochondrial membrane energy gradient, I hypothesized that endotoxin increases the mitochondrial transmembrane potential. Endotoxin treatment (500 micrograms/kg intraperitoneally 48 h earlier) increased the hepatocyte mitochondrial transmembrane potential as determined by two separate methods: the intramitochondrial sequestration of triphenylmethylphosphonium (electrical potential or delta psi) and the fluorescence intensity of the hepatocyte mitochondria when stained with rhodamine-123 and examined by confocal microscopy. These findings suggest that endotoxin treatment increased the total mitochondrial membrane potential per hepatocyte. In parallel, endotoxin treatment increased the fluorescence intensity of hepatocyte mitochondria after staining with 10-N-nonyl-acridine orange, a dye that binds to the mitochondrial inner membrane independently of the transmembrane potential. This suggests that an increase in mitochondrial inner membrane mass is responsible for the net increase in inner membrane potential per cell following endotoxin pretreatment. These findings complement previous studies in which endotoxin treatment increased the mitochondrial-specific antioxidant Mn-SOD and support the more recent finding that endotoxin treatment also increased nonenzymatic scavenging of superoxide by lung mitochondria. Taken, together, these observations suggest that mitochondrial biogenesis, and the subsequent increase in both enzymatic and nonenzymatic scavenging of superoxide anion, is a central feature of endotoxin-mediated tolerance to oxidative stress.

It is commonly accepted that mitochondria undergo major changes early during the apoptotic process and that these alterations are critical for the death/life decision. Here we report that Jurkat T cell leukemia cells exhibit a perturbed incorporation of potential-sensitive fluorochromes. After 6 h of CD95/Fas/APO-1 crosslinking, a significant fraction of still normal-sized Jurkat cells exhibit a decreased incorporation of three different cationic lipophilic dyes commonly used for the quantitation of the mitochondrial transmembrane potential (deltapsi(m)): DiOC6(3), chloromethyl-X-rosamine, and tetramethylrhodaminemethylester. In contrast, upon induction of apoptosis, cells tend to exhibit an increase in the fluorescence obtained with rhodamine 123. The increased rhodamine 123 fluorescence into cells undergoing apoptosis is not affected by labeling in the presence of the protonophore m-chlorophenylhydrazone and thus cannot be attributed to a change in the deltapsi(m). Six hours after CD95 ligation no changes are found among normal-sized cells in the incorporation of mitotracker green and nonylacridine orange, which both measure mitochondrial mass. However, a fraction of cells exhibit an increased staining with the Apo2.7 antibody which detects a mitochondrial antigen generated during apoptosis. These findings underline the importance of using adequate fluorochromes for the quantitation of mitochondrial changes occurring during early apoptosis. Moreover, they cast doubts on those studies that, using rhodamine 123, hypothesized that apoptosis would be associated with a stable or increased deltapsi(m).

9-Aminoacridine carboxamide derivatives studied here form with DNA intercalative complexes which differ in the kinetics of dissociation. Inhibition of total RNA synthesis catalyzed by phage T7 and Escherichia coli DNA-dependent RNA polymerases correlates with the formation of slowly dissociating acridine-DNA complex of time constant of 0.4-2.3 s. Their effect on RNA synthesis is compared with other ligands which form with DNA stable complexes of different steric properties. T7 RNA polymerase is more sensitive to distamycin A and netropsin than the E. coli enzyme while less sensitive to actinomycin D. Actinomycin induces terminations in the transcript synthesized by T7 RNA polymerase. Despite low dissociation rates of DNA complexes with acridines and pyrrole antibiotics no drug dependent terminations are observed with these ligands.

In this preliminary study linear and branched oligosaccharides, derivatized with 2-aminoacridone, have been separated by reverse-phase high-performance liquid chromatography, and detected by positive-ion electrospray mass spectrometry. Tandem Mass spectra of the protonated carbohydrate analytes gave simple fragmentation patterns from which sequence information could be readily obtained.

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is a neurotoxin, which can damage dopaminergic neurons. It causes symptoms resembling those observed in patients suffering from Parkinson's disease, and hence this toxin is widely used in studies on animal models of this disorder. Mutagenicity of MPTP was also reported by some authors, but results obtained by others suggested that this compound is not mutagenic. Interestingly, those contrasting results were based on the same assay (the Ames test). Therefore, we aimed to test MPTP mutagenicity by employing a recently developed Vibrio harveyi assay, which was demonstrated previously to be more sensitive than the Ames test, at least for some mutagens. We found that MPTP showed a significant mutagenic activity. Moreover, MPTP mutagenicity was attenuated by methylxanthines, compounds that are known to form complexes with aromatic mutagens.

The cytotoxic activity, susceptibility to thiol activation and ability of eight 1-nitroacridine derivatives to stabilize the topoisomerase I-DNA cleavable complex, were compared. Among the acridines tested three compounds exhibited high ability to stabilize the cleavable complex. This ability was correlated with susceptibility to thiol activation as well as with cytotoxic activity. Our results suggest that 1-nitroacridine-DNA adducts interfering with topoisomerase I action may contribute to the lethal effects of some 1-nitroacridine derivatives.

2-Aminoacridone (2-AMAC) has been used to derivatize mixtures of N-linked oligosaccharides released from alpha(1)-acid glycoprotein and immunoglobulin G. In each case, the HPLC profile obtained for the derivatized glycans was compared to that obtained after digestion with sialidase and a two-enzyme array system made up of sialidase and alpha-fucosidase, prior to derivatization by 2-AMAC. These studies are rapid and provide a wealth of preliminary information about the degree of sialylation and core fucosylation in the corresponding parent glycans. Moreover, collection of glycans from one single injection has provided enough material for molecular weight determination by MALDI-MS analysis. In this study we have also carried out limited MS-MS studies on enriched fractions of 2-AMAC-glycans using a nanospray orthogonal quadrupole time-of-flight mass spectrometer.

In the present study, the interactions of imidazoacridinones, C-1311 and C-1371 with human erythrocyte membranes were examined by electron spin resonance spectroscopy (ESR). It was observed that both compounds C-1311 and C-1371 preferentially bind to proteins producing the conformational changes of erythrocyte membrane skeletal proteins.

We outline simple methodology for the rapid and selective analysis of 2-aminoacridone (2-AMAC) derivatised oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry. This involves the addition of small amounts of lithium chloride to the matrix before evaporation of solvent and crystallization. Signals mainly attributed to proton, sodium and potassium adducts are suppressed to a great extent, and a single signal due to (M + Li)+ is observed. This technique is rapid and is most useful for the direct analysis of complex glycan mixtures, after derivatization with 2-aminoacridone and without separation of the individual components.

A series of N -monoalkylsubstituted 1,2,3,4-tetrahydro-9-aminoacridines have been prepared after modelling simulation of the AChE–inhibitor complex. Molecular modelling has predicted a number of hydrophobic residues to be involved in the catalytic mechanism of this interaction between the binding sites of AChE and this series of aminoacridines. In these compounds the acridine moiety becomes sandwiched between the rings of PHE330 and TRP84. In particular, the alkyl chain shows the important role of aromatic groups as binding sites. Their in vitro inhibitory properties (enzyme from Electrophorus electricus ) confirm the aromatic groups as a general and significant characteristic of the mechanism of AChE inhibition.

Based on the oxidation of acridine yellow by permanganate in basic medium, a new chemiluminescence system was developed for the sensitive determination of some important drugs. The remarkable inhibiting effect of fluvoxamine, ceftriaxone and isoniazid on this reaction was applied to their detection. A possible mechanism was proposed for this system based on chemiluminescence emission wavelengths and experimental observations. Under optimum conditions, calibration graphs were obtained for 1 × 10(-9) to 1 × 10(-6) mol/L of fluvoxamine; 2 × 10(-8) to 8 × 10(-6) mol/L of ceftriaxone and 5 × 10(-8) to 4 × 10(-5) mol/L of isoniazid. This proposed method was satisfactorily used in the determination of these drugs in pharmaceutical samples and human urine and serum.

A novel synthesis of new acridines via rearrangement of in situ generated indazol-3-ylidenes is developed.

Imidazoacridinone 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) is an antitumor inhibitor of topoisomerase II and FMS-like tyrosine kinase 3 receptor. In this study, we describe the unique sequence of cellular responses to C-1311 in human non-small cell lung cancer (NSCLC) cell lines, A549 and H460. In A549 cells, C-1311 (IC80 = 0.08 µM) induced G1 and G2/M arrests, whereas H460 cells (IC80 = 0.051 µM) accumulated predominantly in the G1 phase. In both cell lines, cell cycle arrest was initiated by overexpression of p53 but was sustained for an extended time by elevated levels of p21. Despite prolonged drug exposure (up to 192 hours), no apoptotic response was detected in either cell line. Instead, cells developed a senescent phenotype and did not resume proliferation even after 2 weeks of post-treatment, indicating that C-1311-triggered senescence was permanent. When cell cycle arrest was evident but there were no signs of senescence, C-1311 significantly induced autophagic cells. Pharmacological inhibition of autophagy by 3-methyladenine profoundly reduced the senescent phenotype and slightly sensitized cancer cells to C-1311 by increasing cell death, suggesting a link between both autophagy and senescence. However, a small interfering RNA-mediated knockdown of the autophagy-associated Beclin 1 and ATG5 genes attenuated but failed to block development of senescence. Taken together, our studies suggest that in NSCLC, a C-1311-induced senescence program is preceded and corroborated but not exclusively determined by the induction of autophagy.

On deprotonation, 1-arylindazolium salts form 1-arylindazol-3-ylidenes which rearrange spontaneously via ring cleavage, ring closure and subsequent proton transfer to substituted 9-aminoacridines. By contrast, the N-heterocyclic carbene of 2-phenylindazolium cannot rearrange similarly and was trapped by sulfur.

The goal of this study was to provide a reasonable assessment of how probe substrate selection may impact the results of in vitro aldehyde oxidase (AO) inhibition experiments. Here, we used a previously studied set of seven known AO inhibitors to probe the inhibition profile of a pharmacologically relevant substrate N-[(2-dimethylamino)ethyl]acridine-4-carboxamide (DACA). DACA oxidation in human liver cytosol was characterized with a measured V(max) of 2.3 ± 0.08 nmol product · min(-1) · mg(-1) and a K(m) of 6.3 ± 0.8 µM. The K(ii) and K(is) values describing the inhibition of DACA oxidation by the panel of seven inhibitors were tabulated and compared with previous findings with phthalazine as the substrate. In every case, the inhibition profile shifted to a much less uncompetitive mode of inhibition for DACA relative to phthalazine. With the exception of one inhibitor, raloxifene, this change in inhibition profile seems to be a result of a decrease in the uncompetitive mode of inhibition (an affected K(ii) value), whereas the competitive mode (K(is)) seems to be relatively consistent between substrates. Raloxifene was found to inhibit competitively when using DACA as a probe, and a previous report showed that raloxifene inhibited uncompetitively with other substrates. The relevance of these data to the mechanistic understanding of aldehyde oxidase inhibition and potential implications on drug-drug interactions is discussed. Overall, it appears that the choice in substrate may be critical when conducting mechanistic inhibition or in vitro drug-drug interactions prediction studies with AO.

The B3LYP method with 6-31G* basis set was used to predict the geometries of five 9-aminoacridines (9-AA 1(a–e)), DNA base pairs, and respective complexes. Polarizabilities, charge distribution, frontier molecular orbital (FMO), and dipole moments were used to analyze the nature of interactions that allow reasonable drug diffusion levels. The results showed that charge delocalization, high polarizabilities, and high dipole moments play an important role in intermolecular interactions with DNA. The interactions of 9-AA 1(a–e) with GC are the strongest. 9-AA 1(d) displayed the strongest interaction and 9-AA 1(b) the weakest.

Persistent hole-burning spectra of protonated acridine, 9-aminoacridine and proflavine doped in polyvinyl alcohol films have been studied to examine how the functional group affects chromophore-glass interactions. Proton transfer between the chromophore and the matrix upon electronic excitation is proposed to be the mechanism for the hole burned spectra of the three molecules studied. The high resolution of the spectrum of 9-aminoacridine is attributed to reduction of the electron-phonon coupling strength when substituting an amino group to the C(9) position of acridine. However, when substituting two amino groups to the C(3) and C(6) positions of acridine, significant increase of the electron-phonon coupling strength decreases the spectral resolution of proflavine.

In the search for chemical probes of DNA conformations, we report an efficient synthesis of new Troger's Base analogs derived from polyfunctionalized aminoacridines treated with a stoechiometric amount of formaldehyde in trifluoroacetic acid. For the more sensitive aminoacridines, the Troger's Bases were obtained by nucleophilic substitution of the chloro group of the “pre-formed” corresponding Troger's Base.

We have studied the actions of C-1311, an imidazoacridinone analogue with potent in vivo antitumour activity, against a human tumour line (HeLa S3), in an examination of the events associated with the lethality of this agent. Continuous exposures (24 h) induced complete G2 arrest, although the concentration range of this effect was narrow, with elevation of the drug level inducing additional and increasing impediment to S-phase transit. Acute treatments (3 h) revealed that cells exposed to drug levels, which first induced persistent G2 arrest (0.5 microgram ml-1), subsequently died from this compartment, while doses exceeding these levels (1.0 microgram ml-1), paradoxically, did not cause the same extensive cell death. We explain our findings on the proposition that this particular mode of cell death is dependent upon inappropriate activation of the primed mitotic machinery-specifically the hyperphosphorylated p34cdc2/cyclin B complex-assembled within G2, but that impediment to genomic replication at higher doses inhibits assembly of this complex, and hence prevents cell death. Our results demonstrate that high dose does not necessarily correlate with increased cell death, while at the same time providing further evidence for the importance of events normally associated with the G2/M transition in DNA damage-induced tumour cell death. Images Figure 7

A highly purified and monodisperse preparation of proton-translocating F1Fo-ATPase from bovine heart mitochondria is an assembly of 16 unlike polypeptides. This preparation has been reconstituted in the presence of various detergents into unilamellar phospholipid vesicles. Incorporation of the enzyme into vesicles increases the ATP hydrolase activity of the enzyme by 10–20-fold, depending on the detergent, and the highest activities of ATP hydrolysis, 70 units/mg, were obtained by reconstitution from dodecylmaltoside or CHAPS. This activity is mostly sensitive to inhibitors that act on the Fo membrane sector of the complex. From the quenching of the pH-sensitive probe, 9-amino-6-chloro-2-methoxyacridine, it was shown that the reconstituted enzyme was able to form a transmembrane proton gradient in an ATP-dependent manner. By co-reconstitution of the enzyme with bacteriorhodopsin, it was demonstrated that in the presence of a light-induced proton gradient the enzyme can synthesize ATP from ADP and phosphate. Therefore, the characteristic biological functions of the F1Fo-ATPase in mitochondria have been demonstrated with the purified enzyme. Thus, in terms of both its physical and biochemical properties, the purified enzyme fulfils important pre-requisites for formation of two- and three-dimensional crystals.

Imidazoacridinones are a new class of highly potent antineoplastic agents synthesised at the Technical University of Gdansk. The pharmacophoric alkyldiamine group, which is also present in anthracenediones (e.g. ametantrone, mitoxantrone), has been shown to be responsible for their antineoplastic activity. In view of their chemical similarity to anthracenediones, we anticipated that the imidazoacridinones would have a mechanism of action similar to that of these agents and that this would be reflected by a similar influence on cell-cycle progression. Flow cytometry was used to monitor the effect of three derivatives of imidazoacridinone (C-1263, C-1310 and C-1311) on L1210 cell cycle traverse at concentrations ranging from 0.01 to 0.9 microgram/ml, corresponding to their 50% and 90% effective concentrations (EC50 and EC90 values), over times of drug treatment ranging from 1 to 48 h. The results demonstrate that all of the compounds produced a similar effect, inducing preferential and complete arrest (accumulation) of cells in the G2 phase of the cell cycle (i.e. G2 block). The kinetics of the induction of G2 arrest were dependent on both the dose and the duration of treatment. Cell-cycle arrest was reversible for up to about 3 h of treatment, being quite irreversible at longer incubation times. Microscopic inspection of cells performed in parallel with flow cytometry confirmed that imidazoacridinones induced a G2, not a G2/M, block.

2-Methoxy-6-chloro-9-aminoacridine has been coupled via a polymethylene linker to various positions of an oligonucleotide chain: the 3'-position, using a new universal support, the 5'-position, and both 5'- and 3'-positions via a phosphate. The intercalating agent was also linked to the oligonucleotide chain via an internucleotide phosphorothiolate. The mixture of diastereoisomers was obtained as well as each pure Rp and Sp isomer. Finally, the acridine moiety was introduced to the 5-position of the deoxyuridine. The binding properties of these oligonucleotide-acridine conjugates with their DNA counterparts have been studied by absorption spectroscopy.

Capillary electrophoresis and laser-induced fluorescence detection have been used to fingerprint the 2-aminoacridone derivatives of complex glycans released from bovine fetuin and human IgG monoclonal antibodies. The utility of this method in distinguishing between N- and O-linked oligosaccharides and in determining the presence of sialic acid residues in glycan mixtures at an early stage of analysis has been demonstrated.

Novel imidazoacridinone derivatives, C1310 and C1311, have been evaluated for their potential to inhibit tumour cell growth in vitro and in vivo. A cell line panel, including seven human and murine colon carcinoma cell lines and three in vivo models, was used. The compounds were found to be potent inhibitors of tumour cell growth with IC50 values ranging between 10 nM and 2 microM in human colon cancer cell lines. Statistically significant tumour growth delay (P < 0.01) was observed after a single intraperitoneal (i.p.) dose of C1311 (100 mg kg-1 body weight) in MAC15A, MAC29 murine and HT29 human adenocarcinomas of the colon. Rapid accumulation of fluorescence of both C1310 and C1311 was seen in the nuclei of HT29 human colon tumour cells in culture. C1311 was also found to bind into calf thymus DNA as shown by spectrophotometric titration and thermal denaturation and to cause early inhibition of thymidine incorporation in HT29 cells in vitro. The results of this study suggest that C1311 should be considered as a candidate for clinical development. Images Figure 3

The fluorophore 2-aminoacridone has been used to label a number of branched oligosaccharides previously released from various glycoproteins. Complex glycans were derivatized via their reducing end with this fluorophore by a Schiff′s base mechanism followed by reduction to a secondary amine using sodium cyanoborohydride. This process of derivatization was carried out efficiently and in a nonselective manner over a period of 30 min at 90°C. The resulting derivatives were separated with high resolution by capillary electrophoresis using borate buffer, containing taurodeoxycholate, as the separation buffer. The method described does not require the removal of sialic acid residues prior to derivatization, so that treatment of glycans with N -acetyl neuraminidase provided useful and additional structural information. Attempts have also been made to relate the electrophoretic mobility of branched oligosaccharides with their molecular volume.

1 A class of compounds, 9-aminoacridines, have long been known to be reversible inhibitors of acetyl cholinesterase (AChE - EC 3.1.1.7), the most familiar of which is 9-amino-1,2,3,4-tetrahydroacridine (Tacrine). 2 A novel aminoacridine was synthesised: - 2-tertiary butyl-9-amino-1,2,3,4-tetrahydroacridine (2tBuTHA). 3 In vitro comparisons of the acetylcholinesterase inhibitory potential and neurotoxicity compared to Tacrine were performed using a chemically differentiated neuroblastoma cell line (Neuro 2A). 2tBuTHA, but not Tacrine, was cytotoxic to the neural cell following 20 h exposure, despite being the least potent AChE inhibitor (IC80 AChE 12.53 μM +/- 1.14 s.e.m., Neutral Red Uptake IC50 9.53 μM +/- 0.98 s.e.m., MTT Reduction IC80 14.6 μM +/- 1.43 s.e.m.). 4 In vivo studies used a novel application of a five arm radial maze to assess neuropharmacological effects on working memory in control and Scopolamine (1 mg kg-1 i.p.) treated mice. There was an impairment of short term cognitive function with 2tBuTHA (15 mg kg -1 i.p.), but not Tacrine (10 mg kg-1 i.p.) which improved the Scopolamine deficit as expected. 5 This combined in vitro and in vivo data infers a neuro toxic property for the novel compound 2tBuTHA, a close structural analogue of Tacrine.

The article reviews evidence for the efficacy of the aminoacridines (tacrine, velnacrine) in patients with probable Alzheimer's disease (AD). The discussion revolves around the question of the clinical relevance of the treatment effects observed. Other questions discussed deal with the heterogeneity of the patient population having AD, the size of the potential reponder group and the limitations caused by safety problems of these compounds. The conclusion is that the effects of the aminoacridines appear to have clinical relevance in a subgroup of patients with AD. These compounds therefore truly deserve the label of ‘first-generation AD agents’. But further basic and clinical research is needed to identify the responder group and predict response.

A method is presented to assess in vivo in transparent tissues the leukocyte subtypes that roll in microvessels. In nine rabbits anesthetized with ketamine-xylazine, leukocyte nuclei were stained in situ with acridine yellow (3 mg/kg i.v. for 5 min). Intravital fluorescence video microscopy in 24 mesenteric venules (17–29 microns, median 21) indicated labeling of all rolling leukocytes. On the basis of the shape of their nucleus, 67–100% (median 89) could be classified unequivocally (13366 cells analyzed, median 77) as polymorphonuclear (PMN, i.e., granulocytes) or monomorphonuclear (lymphocytes and monocytes). Of these classified cells, 94–100% were PMNs (median 100, including 1 stray value of 69%). This PMN percentage was independent of the level of leukocyte rolling (2–36/min, median 14), vessel diameter, flow velocity (0.5–2.5 mm/s), or duration of the experiment (< 6 h). The dye had no significant influence on hemodynamic parameters, systemic leukocyte counts (1.5–7.8 x 10(9)/l), or in vitro differentiation pattern (27–38% granulocytes, 0–2% monocytes, 61–71% lymphocytes). In conclusion, our method demonstrated that the leukocytes that roll in postcapillary venules of the exteriorized rabbit mesentery are almost exclusively granulocytes.

There is increasing evidence that the expression level of drug metabolic enzymes affects the final cellular response following drug treatment. Moreover, anti-tumour agents may modulate enzymatic activity and/or cellular expression of metabolic enzymes in tumour cells. We have investigated the influence of CYP3A4 overexpression on the cellular response induced by the anti-tumour agent C-1311 in hepatoma cells. C-1311-mediated CYP3A4 activity modulation and the effect of CYP3A4 overexpression on C-1311 metabolism have also been examined. With the HepG2 cell line and its CYP3A4-overexpressing variant, Hep3A4, experiments involving DAPI staining, cell cycle analysis, phosphatidylserine externalisation and senescence-associated (SA)-β-galactosidase expression, were used to monitor the effects of C-1311 exposure. C-1311 cellular metabolism and CYP3A4 activity were investigated by high-performance liquid chromatography. C-1311 metabolism was very low in both hepatoma cell lines and slightly influenced by CYP3A4 expression. Interestingly, in HepG2 cells, C-1311 was an effective modulator of CYP3A4 enzymatic activity, being the inhibitor of this isoenzyme in Hep3A4 cells. Cell cycle analysis showed that HepG2 cells underwent a rather stable G(2) /M arrest following C-1311 exposure, whereas CYP3A4-overexpressing cells accumulated only slightly in this compartment. C-1311-treated cells died by apoptosis and necrosis, whereas surviving cells underwent senescence; however, these effects occurred faster and more intensely in Hep3A4 cells. Although CYP3A4 did not influence C-1311 metabolism, changes in CYP3A4 levels affected the C-1311-induced response in hepatoma cells. Therefore, inter-patient differences in CYP3A4 levels should be considered when assessing the potential therapeutic effects of C-1311.