Christian Auclair

Multimodal action of antitumor agents on DNA: The ellipticine series

Most cytotoxic anticancer agents interact directly or indirectly with nuclear DNA, the ultimate target for this class of compounds. For a given type of drug both direct and indirect action at the DNA level usually causes various types of interference or damage. This multimodal mechanism of action is well illustrated by antitumor drugs in the ellipticine series which may bind to DNA through intercalation, may undergo covalent binding, may generate oxidizing species, and may interfere with the catalytic activity of topoisomerase II. The antitumor activity of these compounds may, therefore, result from alternative cytotoxic events. The present review summarizes information obtained with ellipticine compounds on the relation between the nature of the drugs action on DNA and their cytotoxic and/or antitumor activity. The occurrence of topoisomerase-mediated DNA cleavage appears to be responsible for antitumor activity. The capability of the drugs to interfere with the action of topoisomerase II requires the presence of an oxidizable phenolic group on their structure. This feature (or a related one) is shared by all antitumor drugs acting on this enzyme.

Antioxidants in Therapy and Preventive Medicine

Twenty years ago, the enzyme superoxide dismutase which uses the superoxide radical anion as its specific substrate was reported. With this discovery was born a new scientific field, in which oxygen, necessary for aerobi c 1 ife on thi s planet, had to be cons i dered also in terms of its toxicity and stresses. This stimulated the search for knowledge of active oxygen species in biology and medicine. Superoxide and other reactive oxygen species are now implicated in many disease processes. Major advances have been achieved during these past years with respect to free radical generation and mechanisms of free radical action in causing tissue injury. In parallel, the possibil ity of influencing free radical related disease processes by antioxidant treatment was studied in various in vitro and in vivo systems. This was the unique theme of a conference organized in Paris by the Society for Free Radical Research (December 9-10, 1988) which brought together experts from basic sciences and clinicians in order to evaluate the current status of antioxidant therapy. The conference emphasized fundamental processes in antioxidant action. Among the major topics were superoxide dismutase (SOD) and low molecular weight substances with such activity, called SOD mimics. Other antioxidant enzymes were also considered. Antioxidant vitamins, in particular vitamins E and C, other naturally occurri ng antioxidants and vari ous synthet i c antioxidants were included in the presentations as there is now a rapidly developing series of compounds with potentially interesting clinical applications.

Superoxide dismutase assay using alkaline dimethylsulfoxide as superoxide anion-generating system.

Alkaline dimethylsulfoxide as a superoxide anion-generating system in association with cytochrome c as a superoxide anion-indicating scavenger has been used to develop a new assay for superoxide dismutase. The assay is sensitive (one unit of enzymatic activity is provided by 110 ng of purified copper-containing superoxide dismutase) and highly specific. The nature of this system prevents the usual interferences and its simplicity allows for multiple, rapid measurements of superoxide dismutase activity in biological preparations using either normal or automated procedures.

Oxidative Stress, Cell Activation and Viral Infection

DNA glycosylases involved in the repair of oxidized bases in Escherichia coli.- Potential role of protein oxidation and proteasome in antigen processing.- Involvement of hydrogen peroxide in the actions of TGF ?1.- Oxidative stress and growth factor-mediated signal transduction.- Cellularly generated active oxygen species as signals in the activation of tumour cell growth.- Effect of oxygen radicals on the IL-1 production by monocytes and IL-2 receptor expression in lymphocytes during primary and secondary immunodeficiency.- The effect of reactive oxygen species (ROS) on human T and B lymphoid cells.- Leukocyte adhesion and endothelial cytokine production in hypoxia/reoxygenation.- Effects of antioxidants on IL-6 secretion induced by IL-1 in human cultured lung fibroblasts. Involvement of NF kB.- Hydrogen peroxide-induced synthesis of the 32kDa stress protein (HO-1) in endothelial cells is serum dependent.- The antioxidant effects of glutathione and ascorbic acid.- Structural consequences of NF-kB inhibition by natural antioxidants: ?-lipoic acid and vitamin E.- d-?-tocopherol and cell proliferation.- Effects of intracellular redox status on cellular regulation and viral infection.- Mechanisms of regulation of cell growth by cytokines of the immune system.- Oxidization of human low density lipoproteins measured by Laser Doppler electrophoresis.- Selective modulation of brain antioxidant defense capacity by genetic or metabolic manipulations.- Protective effect of Poly(A)-poly(U) against immune oxidative injury. Role of thiols released by activated macrophages.- Reactive oxygen, antioxidants, and autotoxicity in viral diseases.- Redox control of gene expression by eukaryotic transcription factors NF-kB, AP-1 and SRF/TCF.- Redox mechanisms in Tcell activation.- Implication of oxydative phenomena in T cell activation.- Intracellular damages induced by singlet oxygen are signals for HIV-1 reactivation.- Abnormal redox regulation in HIV infection and other immunodeficiency diseases.- Influence of redox status of lymphocytes and monocytes on HIV expression and immune functions. Evaluation in vitro of antioxidant molecules as potential anti-HIV therapy.- Place for an antioxidant therapy in human immunodeficiency virus (HIV) infection.- Prevention of early cell death in peripheral blood lymphocytes of HIV infected individuals by an antioxidant: N-Acetyl-cysteine.- Alcanes measurements in human immunodeficiency virus infection.- Plasma antioxidant status (selenium, retinol and ?-tocopherol) in HIV infection.- Author index.- Keyword index.

The formation of superoxide radical anions by a reaction between O2, OH− and dimethyl sulfoxide

Abstract Addition of OH − to air saturated dimethyl sulfoxide leads to the formation of the superoxide radical anion, as shown directly by electron paramagnetic resonance and ultra violet spectroscopy, and indirectly by superoxide dismutase inhibitable cytochrome c reduction. Superoxide production is related inversely to the water concentration of the dimethyl sulfoxide and solutions obtained are stable for up to three days. Reaction mechanisms are suggested and results are discussed in the light of the many uses of dimethyl sulfoxide as a solvent in both chemistry and biology.

Clastogenic inosine nucleotide as components of the chromosome breakage factor in scleroderma patients

In the present study, we attempted to identify the chemical nature of the clastogenic factor (CF) from patients with progressive systemic sclerosis (scleroderma). Computerized mass spectrometry of clastogenic fractions obtained by HPLC of plasma ultrafiltrates detected molecular peaks compatible with inosine triphosphate and inosine diphosphate (ITP and IDP). The concomitant detection of IDP, together with ITP, and the absence of these peaks in nonclastogenic fractions and corresponding control fractions are arguments in favor of a biological relevance of these observations. The most important confirmation came from the clastogenic effect of commercial ITP and IDP added to the culture medium of the test cultures. The induction of chromatid type damage by these substances in lymphocytes exposed in the G0 phase of their cell cycle and the prevention of this damage by superoxide dismutase are analogous to the observations with CF.

A revised structure of the antitumor drug elliptinium — Amino(acid) adducts

Using simple organic reactions and spectrometric techniques (nuclear magnetic resonance and mass spectrometry), we propose a revised structure of the adducts obtained through the H2O2-peroxidase oxidation of the antitumor drug ellipticinium and aliphatic amino(acid) compounds. These derivatives display an oxazole ring structure between the O(9)- and N(10)- atoms.

Inhibition of the in vitro integration of Moloney murine leukemia virus DNA by the DNA minor groove binder netropsin.

In search of potential inhibitors of integration of retroviral DNA into host cells genome, we have investigated the effect of the external DNA binder netropsin on the in vitro insertion of long terminal repeat (LTR) ends of Moloney murine leukemia virus (M.MuLV) as catalysed by integrase purified from baculovirus strain expression vector. In agreement with the preferential binding of netropsin to A+T rich sequences, footprinting experiments have shown that this drug selectively binds to the 5-TTTCAT LTR end sequence which is included in the DNA binding site of integrase. This feature results in the potent inhibition of both reactions involved in the insertion process, namely, nucleolytic cleavage and strand transfer. The relation between netropsin binding to A+T rich region of M.MuLV LTR end and inhibition of insertion is strongly suggested from the inability of the drug to inhibit the insertion of HIV U3 LTR end which displays a G+C rich sequence. Selective inhibition of integration of viral DNA appears to be feasible using drugs recognizing LTR end sequences.

A Molecular Mechanics and Dynamics Study of Alternate Triple-Helices Involving the Integrase-Binding Site of the HIV-1 Virus and Oligonucleotides Having a 3′-3′ Internucleotide Junction

Triple helix formation by oligonucleotides can be extended beyond polypurine tracts with the help of specially designed linkers. In this paper we focus our attention on the integrase-binding site of the HIV-1 virus located on the U5 LTR end which contains two adjacent purine tracts on opposite strands. Two alternate triple helices with a 3-3 junction in the third strand are considered: 5-GGTTTTp3-3pTGTGT-5 and 5-GGAAAAp3-3pAGAGA-5 The structural plausibility of these triplexes is investigated using molecular mechanics and dynamics simulations, both in vacuo and in aqua. The non-isomorphism of the triplets in the GpT steps in the first sequence, gives rise to non canonical conformations in the torsion angles, hydration appears to be crucial for this triplex. Sugar puckers are predominantly South during in vacuo simulations while they turn East in aqua. In the simulation in aqua the triplexes are shrouded by an hydration shell, however, we have not been able to detect any permanent hydrogen bond bridge between DNA and water. The solvation of ions as well as their radial distribution, appear to be relatively well behaved despite the artifacts known to be generated by the simulation procedure. The experimental feasibility of these structures is discussed.

Binding of Net-Fla, a netropsin-flavin hybrid molecule, to DNA: molecular mechanics and dynamics studies in vacuo and in water solution.

We have studied the binding of the hybrid netropsin-flavin (Net-Fla) molecule onto four sequences containing four A. T base pairs. Molecular mechanics minimizations in vacuo show numerous minimal conformations separated by one base pair. 400 ps molecular dynamics simulations in vacuo have been performed using the lowest minima as the starting conformations. During these simulations, the flavin moiety of the drug makes two hydrogen bonds with an amino group of a neighboring guanine. A 200 ps molecular dynamics simulation in explicit water solution suggests that the binding of Net-Fla upon the DNA substrate is enhanced by water bridges. A water molecule bridging the amidinium of Net-Fla to the N3 atom of an adenine seems to be stuck in the drug-DNA complex during the whole simulation. The fluctuations of the DNA helical parameters and of the torsion angles of the sugar-phosphate backbone are very similar in the simulations in vacuo and in water. The time auto-correlation functions for the DNA helical parameters decrease rapidly in the picosecond range in vacuo. The same functions computed from the water solution molecular dynamics simulations seem to have two modes: the rapid mode is similar to the behavior in vacuo, and is followed by a slower mode in the 10 ps range.