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1.
Biol Cell ; 91(8): 597-604, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10629939

ABSTRACT

The effect of controlled and reversible perturbation of the electrochemical gradient on the structural changes of mitochondrial DNA has been studied in living cells by fluorescence microscopy. Electrochemical gradient perturbations were induced by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and quantified by measuring the mitochondrial membrane potential using tetramethyl rhodamine methyl ester. Under our experimental conditions, we have shown that ethidium fluorescence was mainly due to ethidium molecules intercalated in mtDNA. Ethidium fluorescence variations have been used to probe DNA structural changes. This showed that: i) electrochemical gradient perturbations induced mtDNA structural change; ii) this change was readily reversible following a total but short collapse of the electrochemical gradient; iii) in contrast, a short and weak perturbation of the electrochemical gradient stabilized the mtDNA structural change; and iv) the degree of weak depolarization varied from cell to cell, showing the necessity of studying the effect of energetic perturbations at the level of an individual cell.


Subject(s)
DNA, Mitochondrial/chemistry , DNA, Mitochondrial/physiology , Electrochemistry , Mitochondria/physiology , Animals , Atractyloside/pharmacology , Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone/pharmacology , Chlorocebus aethiops , Enzyme Inhibitors/pharmacology , Ethidium , Fluorescent Dyes , Ionophores/pharmacology , Kinetics , Membrane Potentials/drug effects , Membrane Potentials/physiology , Microscopy, Fluorescence , Nucleic Acid Conformation , Oligomycins/pharmacology , Rhodamines , Vero Cells
2.
Microsc Res Tech ; 36(4): 287-95, 1997 Feb 15.
Article in English | MEDLINE | ID: mdl-9140928

ABSTRACT

Low light level fluorescence microscopy studies have been carried out on MCF7-P human mammary tumor cells to localize the intracellular distribution of two new anticancer drugs, Pazelliptine and Intoplicine, which are currently under clinical evaluation. These two molecules are thought to act at the nuclear level, through DNA topoisomerase interactions. Because fluorescence of these compounds appears strongly quenched by intercalation in double strand DNA, secondary ion mass spectrometry (SIMS) imaging was used to check the presence of the drugs in the nuclear compartment. In spite of chemical structure similitudes, pazelliptine and intoplicine appear to be distributed in quite different ways within the cells. Incubation for 1 and 24 hours also allowed us to bring to light strong differences in the distribution kinetics. Pazelliptine quickly enters into the nucleoli but is no longer present in the nucleus after 24 hours incubation. Intoplicine was not detected by fluorescence in the nucleus, however SIMS microscopy allowed us to show its accumulation within this cellular compartment as a function of time of exposure. This study shows the complementarity of fluorescence and SIMS microscopies.


Subject(s)
Antineoplastic Agents/pharmacokinetics , Indoles/pharmacokinetics , Isoquinolines/pharmacokinetics , Pyridines/pharmacokinetics , Humans , Mass Spectrometry , Microscopy, Fluorescence , Tumor Cells, Cultured
3.
Biophys J ; 71(5): 2319-28, 1996 Nov.
Article in English | MEDLINE | ID: mdl-8913573

ABSTRACT

Digital-imaging microscopy was used in conditions that allowed the native state to be preserved and hence fluorescence variations of specific probes to be followed in the real time of living mammalian cells. Ethidium bromide was shown to enter into living cells and to intercalate stably into mitochondrial DNA (mtDNA), giving rise to high fluorescence. When the membrane potential or the pH gradient across the inner membrane was abolished by specific inhibitors or ionophores, the ethidium fluorescence disappeared from all mtDNA molecules within 2 min. After removal of the inhibitors or ionophores, ethidium fluorescence rapidly reappeared in mitochondria, together with the membrane potential. The fluorescence extinction did not result from an equilibrium shift caused by leakage of free ethidium out of mitochondria when the membrane potential was abolished but was most likely due to a dynamical mtDNA change that exposed intercalated ethidium to quencher, either by weakening the ethidium binding constant or by giving access of a proton acceptor (such as water) to the interior of mtDNA. Double labeling with ethidium and with a minor groove probe (4',6-diamino-2-phenylindole) indicated that mtDNA maintains a double-stranded structure. The two double-stranded DNA states, revealed by the fluorescence of mitochondrial ethidium, enhanced or quenched in the presence of ethidium, seem to coexist in mitochondria of unperturbed fibroblast cells, suggesting a spontaneous dynamical change of mtDNA molecules. Therefore, the ethidium fluorescence variation allows changes of DNA to be followed, a property that has to be taken into consideration when using this intercalator for in vivo as well as in vitro imaging studies.


Subject(s)
DNA, Mitochondrial/chemistry , Mitochondria/physiology , DNA, Mitochondrial/metabolism , Electrochemistry , Energy Metabolism , Ethidium/chemistry , Fibroblasts , Humans , Indoles/chemistry , Membrane Potentials , Mitochondria/ultrastructure , Nigericin/pharmacology , Nucleic Acid Conformation , Skin/cytology
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