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Disulfiram has been used for decades to treat alcoholism. Its therapeutic effect is thought to be mediated by the irreversible inhibition of aldehyde dehydrogenase. Recent reports have indicated new therapeutic uses of disulfiram, in particular in human cancers. Although the biochemical mechanisms that underlie these effects remain largely unknown, certain enzymes involved in cancer processes have been reported to be targeted by disulfiram. Arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizing enzyme that biotransforms aromatic amine drugs and carcinogens. In addition to its role in xenobiotic metabolism, several studies have suggested that NAT1 is involved in other physiological and/or pathological processes, such as folate metabolism or cancer progression. In this report, we provide evidence that human NAT1 is a new enzymatic target of disulfiram. We found that disulfiram at clinically relevant concentrations impairs the activity of endogenous NAT1 in human cancer cells. Further mechanistic and kinetic studies indicated that disulfiram reacts irreversibly with the active site cysteine residue of NAT1, leading to its rapid inhibition (IC50 = 3.3 ± 0.1 μm and ki = 6 × 104 m−1·min−1).

Mitochondria are dynamic organelles with continuous fusion and fission, the equilibrium of which results in mitochondrial morphology. Evidence points to there being an intricate relationship between mitochondrial dynamics and oxidative phosphorylation. We investigated the bioenergetics modulation of mitochondrial morphology in five control cultured primary skin fibroblasts and seven with genetic alterations of oxidative phosphorylation. Under basal conditions, control fibroblasts had essentially filamentous mitochondria. Oxidative phosphorylation inhibition with drugs targeting complex I, III, IV or V induced partial but significant mitochondrial fragmentation, whereas dissipation of mitochondrial membrane potential (DΨm) provoked complete fragmentation, and glycolysis inhibition had no effect. Oxidative phosphorylation defective fibroblasts had essentially normal filamentous mitochondria under basal conditions, although when challenged some of them presented with mild alteration of fission or fusion efficacy. Severely defective cells disclosed complete mitochondrial fragmentation under glycolysis inhibition. In conclusion, mitochondrial morphology is modulated by DΨm but loosely linked to mitochondrial oxidative phosphorylation. Its alteration by glycolysis inhibition points to a severe oxidative phosphorylation defect.

Eukaryotic cells contain numerous copies of the mitochondrial genome (from 50 to 100 copies in the budding yeast to some thousands in humans) that localize to numerous intramitochondrial nucleoprotein complexes called nucleoids. The transmission of mitochondrial DNA differs significantly from that of nuclear genomes and depends on the number, molecular composition and dynamic properties of nucleoids and on the organization and dynamics of the mitochondrial compartment. While the localization, dynamics and protein composition of mitochondrial DNA nucleoids begin to be described, we are far from knowing all mechanisms and molecules mediating and/or regulating these processes. Here, we review our current knowledge on vertebrate nucleoids and discuss similarities and differences to nucleoids of other eukaryots.

Enlarged or giant mitochondria have often been documented in aged tissues although their role and underlying mechanism remain unclear. We report here how highly elongated giant mitochondria are formed in and related to the senescent arrest. The mitochondrial morphology was progressively changed to a highly elongated form during deferoxamine (DFO)-induced senescent arrest of Chang cells, accompanied by increase of intracellular ROS level and decrease of mtDNA content. Interestingly, under exposure to subcytotoxic doses of H2O2 (200 µM), about 65% of Chang cells harbored elongated mitochondria with senescent phenotypes whereas ethidium bromide (EtBr) (50 ng/ml) only reformed the cristae structure. Elongated giant mitochondria were also observed in TGF β1- or H2O2-induced senescent Mv1Lu cells and in old human diploid fibroblasts (HDFs). In all senescent progresses employed in this study Fis1 protein, a mitochondrial fission modulator, was commonly downexpressed. Overexpression of YFP-Fis1 reversed both mitochondrial elongation and appearance of senescent phenotypes induced by DFO, implying its critical involvement in the arrest. Finally, we found that direct induction of mitochondrial elongation by blocking mitochondrial fission process with Fis1-ΔTM or Drp1-K38A was sufficient to develop senescent phenotypes with increased ROS production. These data suggest that mitochondrial elongation may play an important role as a mediator in stress-induced premature senescence. J. Cell. Physiol. 209: 468–480, 2006. © 2006 Wiley-Liss, Inc.

Mitochondria are essential organelles that are involved in numerous metabolic pathways and produce the major part of intracellular ATP by oxidative phosphorylation. Their ultrastructure was solved in the 1950s by electron microscopic analysis of ultrathin sections. Based on these pioneering studies and on the endosymbiotic origin of mitochondria, cells are often assumed to contain numerous independent mitochondria with a size similar to that of bacteria. However, electron microscopy of thick sections reveals that mitochondria form elongated and branched filaments. Optical microscopy of living cells demonstrates that mitochondrial filaments continuously modify their position and morphology and that they undergo frequent fission and fusion reactions. In this review, we revise the actual knowledge on the ultrastructure, the organization and the dynamics of the mitochondrial compartment. We review recent findings showing that mitochondria exchange molecules by fusion and we present the main proteins involved in mitochondrial fusion and fission reactions. Finally, we discuss the functional and physiological relevance of mitochondrial dynamics.

Disulfiram has been used for decades to treat alcoholism. Its therapeutic effect is thought to be mediated by the irreversible inhibition of aldehyde dehydrogenase. Recent reports have indicated new therapeutic uses of disulfiram, in particular in human cancers. Although the biochemical mechanisms that underlie these effects remain largely unknown, certain enzymes involved in cancer processes have been reported to be targeted by disulfiram. Arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizing enzyme that biotransforms aromatic amine drugs and carcinogens. In addition to its role in xenobiotic metabolism, several studies have suggested that NAT1 is involved in other physiological and/or pathological processes, such as folate metabolism or cancer progression. In this report, we provide evidence that human NAT1 is a new enzymatic target of disulfiram. We found that disulfiram at clinically relevant concentrations impairs the activity of endogenous NAT1 in human cancer cells. Further mechanistic and kinetic studies indicated that disulfiram reacts irreversibly with the active site cysteine residue of NAT1, leading to its rapid inhibition (IC50 = 3.3 +/- 0.1 microM and k(i) = 6 x 10(4) M(-1) x min(-1)).

Mitochondria are almost ubiquitous organelles in Eukaryota. They are highly dynamic and often complex structures in the cell. The mammalian mitochondrial proteome is predicted to comprise as many as 2000-2500 different proteins. Determination of the subcellular localization of any newly identified protein is one of the first steps toward unraveling its biological function. For most mitochondrial proteins, this can now be done relatively easily by cloning a complementary deoxyribonucleic acid of interest in frame with an additional sequence for a fluorescent or nonfluorescent protein tag. Transfection and subsequent visualization, either by direct fluorescence microscopy or by indirect immunofluorescence microscopy, will give the first clue to mitochondrial localization. In combination with a fluorescent "marker" dye, the mitochondrial localization can be confirmed. This chapter describes some of the methods used in determining mitochondrial protein localization, which can also be used to study dynamics of mitochondria or individual mitochondrial proteins or protein complexes.

Mitochondria form a dynamic network in which continuous movement, fusion, and division ensure the distribution and exchange of proteins and deoxyribonucleic acid (DNA). The recent past has seen the identification and characterization of the first proteins governing the organization, function, and dynamics of mitochondria and mitochondrial DNA, and it is predictable that numerous new proteins will require localization and functional characterization in the future. In this chapter, we describe methods for the visualization of mitochondria and mitochondrial activity in cultured mammalian cells to establish the localization or distribution of its components and to study mitochondrial fusion.

Mitochondria are almost ubiquitous organelles in Eukaryota. They are highly dynamic and often complex structures in the cell. The mammalian mitochondrial proteome is predicted to comprise as many as 2000-2500 different proteins. Determination of the subcellular localization of any newly identified protein is one of the first steps toward unraveling its biological function. For most mitochondrial proteins, this can now be done relatively easily by cloning a complementary deoxyribonucleic acid of interest in frame with an additional sequence for a fluorescent or nonfluorescent protein tag. Transfection and subsequent visualization, either by direct fluorescence microscopy or by indirect immunofluorescence microscopy, will give the first clue to mitochondrial localization. In combination with a fluorescent "marker" dye, the mitochondrial localization can be confirmed. This chapter describes some of the methods used in determining mitochondrial protein localization, which can also be used to study dynamics of mitochondria or individual mitochondrial proteins or protein complexes.

Mitochondria are enveloped by two closely apposed boundary membranes with different properties and functions. It is known that they undergo fusion and fission, but it has remained unclear whether outer and inner membranes fuse simultaneously, coordinately or separately. We set up assays for the study of inner and outer membrane fusion in living human cells. Inner membrane fusion was more sensitive than outer membrane fusion to inhibition of glycolysis. Fusion of the inner membrane, but not of the outer membrane, was abolished by dissipation of the inner membrane potential with K+ (valinomycin) or H+ ionophores (cccp). In addition, outer and inner membrane fusion proceeded separately in the absence of any drug. The separate fusion of outer and inner membranes and the different requirements of these fusion reactions point to the existence of fusion machineries that can function separately.

Heteroplasmic mutations of mitochondrial DNA (mtDNA) are an important source of human diseases. The mechanisms governing transmission, segregation and complementation of heteroplasmic mtDNA-mutations are unknown but depend on the nature and dynamics of the mitochondrial compartment as well as on the intramitochondrial organization and mobility of mtDNA. We show that mtDNA of human primary and immortal cells is organized in several hundreds of nucleoids that contain a mean of 2-8 mtDNA-molecules each. Nucleoids are enriched in mitochondrial transcription factor A and distributed throughout the entire mitochondrial compartment. Using cell fusion experiments, we demonstrate that nucleoids and respiratory complexes are mobile and diffuse efficiently into mitochondria previously devoid of mtDNA. In contrast, nucleoid-mobility was lower within mitochondria of mtDNA-containing cells, as differently labeled mtDNA-molecules remained spatially segregated in a significant fraction (37%) of the polykaryons. These results show that fusion-mediated exchange and intramitochondrial mobility of endogenous mitochondrial components are not rate-limiting for intermitochondrial complementation but can contribute to the segregation of mtDNA molecules and of mtDNA mutations during cell growth and division.