Claude Paoletti

A fluorescent complex between ethidium bromide and nucleic acids: Physical—Chemical characterization

Abstract At high salt concentrations, ethidium bromide (EB) is shown to form a fluorescent complex with helical polynucleotides which has a strikingly enhanced fluorescence quantum yield. In this paper we present evidence that formation of this complex (complex I) ia specific for base-paired regions either in DNA, RNA or RNA: DNA hybrids, and that the basis of this specificity is intercalation of the dye between base pairs. At low salt concentrations a second mode of binding EB‡ (as complex II) is observed: it appears to be electrostatic binding of the cationic dye to negatively charged phosphate groups. Complex II can be distinguished readily from complex I by its fluorescence properties. The nature of the DNA binding sites for EB has been investigated by: (a) measurement of their number, and dependence of the binding on pH and salt concentration, as well as on the secondary structure and GC content of the DNA; (b) study of the energy transfer from DNA to EB; (c) studies of competition for DNA between EB and Na+, Mg2+, quinacrine and actinomycin, and (d) characterization of complex I by flow dichroism, thermal melting curves and buoyant density.

A new fluorometric method for RNA and DNA determination

Abstract Previous studies have shown that a dye, ethidium bromide, markedly increases its fluorescence when it binds to double-stranded DNA or double-stranded regions of RNA. It is shown that, in the given conditions, a linear relationship between fluorescence and nucleic acid concentration is obtained. Thus, a new spectrofluorometric method for determining DNA and RNA using this principle is described. This method is specific for nucleic acids, can be used over a wide range of salt concentrations, and has a good sensitivity (0.01 μg/ml of DNA). No biological compounds able to interfere have been found. An adaptation of this method for determining both DNA and RNA in a mixture is also described.

Electrochemotherapy potentiation of antitumour effect of bleomycin by local electric pulses

In cell culture the cytotoxicity of some anticancer drugs, especially bleomycin, can be greatly enhanced by exposing cells to non-cytotoxic electric pulses. Nude or conventional mice bearing subcutaneous transplanted tumours were treated with intramuscular doses of bleomycin followed by local delivery of electric pulses similar to those used in vitro. Tumors were reduced and even eradicated after this electrochemotherapy. Thus the antitumour effects of bleomycin in mice can be considerably potentiated by local electric pulses.

Transient electropermeabilization of cells in culture: Increase of the cytotoxicity of anticancer drugs

The electropermeabilization (EPN) of living cells allows the uptake of non-permeant molecules and can reveal their potential activity on cells without the constraints of the plasma membrane crossing. We decided to compare the cytotoxicity of some anticancer drugs on electropermeabilized (EP) and non-permeabilized (NEP) cultured DC-3F cells exposed to the drugs for a short time. After EPN, the increase in cytotoxicity varies between 1 and more than 700 times, depending on the usual cell uptake pathway of a given drug. The most relevant increase of toxicity was observed with molecules such as netropsin (200-fold) and bleomycin (700-fold) which in ordinary conditions weakly diffuse through the plasma membrane. Only a 3-5-fold increase of the cytotoxicity was observed with lipophilic drugs able to rapidly diffuse through the plasma membrane (actinomycin D, NMHE) both in the case of drug-sensitive and resistant cell strains. This increased toxicity is clearly related to a facilitated uptake because, after electropermeabilization, the effects of melphalan (a drug which enters intact cells via leucine transporters) are not modulated by the external leucine concentration. Thus, EPN enables us to reveal the intrinsic toxicity of hydrophilic molecules which have a limited access to their intracellular targets. We propose that EPN can be used as a novel screening procedure of new cytotoxic molecules which could be modified thereafter in order to facilitate their cellular uptake.

Introduction of definite amounts of nonpermeant molecules into living cells after electropermeabilization: direct access to the cytosol.

The possibility of introducing definite amounts of nonpermeant molecules into electropermeabilized living cells has been approached by quantifying the amounts of Lucifer Yellow (LY; a 457-Da highly fluorescent molecule) and Phytolacca americana (Pokeweed) antiviral protein (PAP; a 30,000-Da ribosome-inactivating protein) retained by the cells after closure of the electric-field-induced transient structures of permeation. Without the electropermeabilization, these two molecules enter the cell only in very small amounts by fluid-phase pinocytosis. Under our experimental conditions, using the NIH 3T3 cells, the intracellular LY concentration can reach a value equivalent to the extracellular concentration and can be regulated by controlling the external concentration. We describe the use of LY in a rapid and efficient test for the determination of the best electrical-shock conditions of other cell lines. After electropermeabilization, PAP is 2 X 10(5) times more cytotoxic. Its toxicity can be detected at external concentrations (10(-11) M) corresponding to less than 10 internalized molecules per electropermeabilized cell. Therefore, after electropermeabilization, the nonpermeant molecules have a direct access to the cytosol and the biological effect of nonpermeant substances can be revealed.

Electrochemotherapy of spontaneous mammary tumours in mice

Electrochemotherapy delivers external electric pulses to the tumour site to induce local potentiation of the antitumour activity of intramuscular injections of bleomycin. C3H/Bi mice with spontaneous mammary carcinomas received weekly injections of 50 micrograms bleomycin followed by electric pulses 30 min later. All the 38 tumours treated exhibited at least a partial regression. 23 complete remissions were observed, 3 of which were cures. One difficulty in assessing the cure rate in this model is that frequent parallel or sequential tumours cause early death. Electrochemotherapy appears similarly efficient in spontaneous tumours as in previously studied transplanted tumours.

Ellipticines as potent inhibitors of aryl hydrocarbon hydroxylase: Their binding to microsomal cytochromes p450 and protective effect against benzo(a)pyrene mutagenicity

Abstract Ellipticine (5,11 -dimethyl-6-H-pyrido[4-3b]carbazole) and its derivatives bind strongly to the oxidized and reduced liver microsomal cytochromes P450 of differently pretreated rats, producing typical difference spectra, with peaks respectively at 428 (ox.) and 445 nm (red.) (with spectral dissociation constants around 10 −6 and 10 −5 M, respectively). The high affinity of ellipticine for microsomes containing a great proportion of cytochrome P448, explains its role of strong inhibitor of benzo(a)pyrene hydroxylase. Accordingly, we found a good correlation between the binding properties of the ellipticines and their inhibitory effect upon: (1) the microsomal formation of water soluble metabolites of benzo(a)pyrene, (2) the covalent binding of reactive metabolites of this hydrocarbon to DNA, and (3) the mutagenic activity of benzo(a)pyrene in the Salmonella typkimurium test.

Characterization of a new class of circular DNA molecules in yeast

Abstract This paper describes the isolation of a pure population of covalently closed circular twisted DNA molecules from yeast. These molecules are homogeneous in size, that is consist of monomers of 2.2μ and of multiple length oligomers of n x 2.2μ. While no data rule out the mitochondrial origin of this DNA, its actual intracellular localization remains unknown; it displays the same buoyant density as the main nuclear DNA and therefore is not the heavy nuclear satellite DNA (γ-DNA described by Moustacchi and Williamson (1966) ); although circular molecules represent only 1 to 5 % of the total DNA, they can be prepared in sizable and reproducible amounts by a method based on the use of mechanical disruption of yeast cells rather than lysis by snail gut juice.

Comparative activity of α- and β-anomeric oligonucleotides on rabbit β globin synthesis: Inhibitory effect of cap targeted α-oligonucleotides

Abstract α-anomeric oligonucleotides are resistant to nucleases and display parallel annealing to RNA complementary sequences. We compared the effect of α- and β-oligonucleotides targeted against various mRNA regions on the rabbit β globin in vitro synthesis. In order to determine the role of RNase H, experiments were performed in both rabbit reticulocyte lysate and wheat germ extract. As expected β-oligonucleotides were found more efficient in wheat germ extract which is rich in RNase H activity and α-oligonucleotide targeted against the initiation codon or downstream had no effect because they do not induce mRNA cleavage by RNase H. However, we report, for the first time, a specific translation inhibition by α-oligonucleotides. This occurs provided they are targeted against the cap region in 5′ of the mRNA.

The hydroxylation of the antitumor agent, ellipticine, by liver microsomes from differently pretreated rats

Abstract The antitumor agent, ellipticine (5,11-dimethyl-6H-pyrido[4-3,b]carbazole) is mainly hydroxylated in position 9 by liver microsomes of differently pretreated rats, this result being in agreement with that obtained previously in vivo. A quick and reliable fluorometric assay, based on the differential fluorescent properties of ellipticine and 9-hydroxyellipticine, is described for the measurement of the 9-hydroxylase activity of different microsomes. This activity exhibits the usual features of the cytochrome-P450-dependent monooxygenases. Control rat liver microsomes exhibit a good affinity for ellipticine (Km = 3 × 10−5 M) but a low specific activity (0.1 nmole min−1 mg protein −1), perhaps related with an excess substrate inhibition. Pretreatment of rats with benzo[a]pyrene or ellipticine enhances the rate of 9-hydroxylation: pretreatment with phenobarbital does not. Metyrapone and 7,8 benzofiavone are poor inhibitors of ellipticine hydroxylation particularly in microsomes from benzo[a]pyreneor ellipticine-pretreated rats.