Rate-limiting step preceding cytochrome c release in cells primed for Fas-mediated apoptosis revealed by analysis of cellular mosaicism of respiratory changes

In the present work, Jurkat cells undergoing anti-Fas antibody (anti-Fas)-triggered apoptosis exhibited in increasing proportion a massive release of cytochrome c from mitochondria, as revealed by double-labeling confocal immunofluorescence microscopy. The cytochrome c release was followed by a progressive reduction in the respiratory activity of the last respiratory enzyme, cytochrome c oxidase (COX), and with a little delay, by a decrease in overall endogenous respiration rate, as measured in vivo in the whole cell population. Furthermore, in vivo titration experiments showed that an approximately 30% excess of COX capacity over that required to support endogenous respiration, found in naive cells, was maintained in anti-Fas-treated cells having lost approximately 40% of their COX respiratory activity. This observation strongly suggested that only a subpopulation of anti-Fas-treated cells, which maintained the excess of COX capacity, respired. Fractionation of cells on annexin V-coated paramagnetic beads did indeed separate a subpopulation of annexin V-binding apoptotic cells with fully released cytochrome c and completely lacking respiration, and a nonbound cell subpopulation exhibiting nearly intact respiration and in their great majority preserving the mitochondrial cytochrome c localization. The above findings showed a cellular mosaicism in cytochrome c release and respiration loss, and revealed the occurrence of a rate-limiting step preceding cytochrome c release in the apoptotic cascade. Furthermore, the striking observation that controlled digitonin treatment caused a massive and very rapid release of cytochrome c and complete loss of respiration in the still respiring anti-Fas-treated cells, but not in naive cells, indicated that the cells responding to digitonin had already been primed for apoptosis, and that this treatment bypassed or accelerated the rate-limiting step most probably at the level of the mitochondrial outer membrane.     

Complementation of mutant and wild-type human mitochondrial DNAs coexisting since the mutation event and lack of complementation of DNAs introduced separately into a cell within distinct organelles.

The rules that govern complementation of mutant and wild-type mitochondrial genomes in human cells were investigated under different experimental conditions. Among mitochondrial transformants derived from an individual affected by the MERRF (myoclonus epilepsy associated with ragged red fibers) encephalomyopathy and carrying in heteroplasmic form the mitochondrial tRNA(Lys) mutation associated with that syndrome, normal protein synthesis and respiration was observed when the wild-type mitochondrial DNA exceeded 10% of the total complement. In these transformants, the protective effect of wild-type mitochondrial DNA was shown to involve interactions of the mutant and wild-type gene products. Very different results were obtained in experiments in which two mitochondrial DNAs carrying nonallelic disease-causing mutations were sequentially introduced within distinct organelles into the same human mitochondrial DNA-less (rho 0) cell. In transformants exhibiting different ratios of the two genomes, no evidence of cooperation between their products was observed, even 3 months after the introduction of the second mutation. These results pointed to the phenotypic independence of the two genomes. A similar conclusion was reached in experiments in which mitochondria carrying a chloramphenicol resistance-inducing mitochondrial DNA mutation were introduced into chloramphenicol-sensitive cells. A plausible interpretation of the different results obtained in the latter two sets of experiments, compared with the complementation behavior observed in the heteroplasmic MERRF transformants, is that in the latter, the mutant and wild-type genomes coexisted in the same organelles from the time of the mutation. This would imply that the way in which mitochondrial DNA is sorted among different organelles plays a fundamental role in determining the oxidative-phosphorylation phenotype in mammalian cells. These results have significant implications for mitochondrial genetics and for studies on the transmission and therapy of mitochondrial DNA-linked diseases.     

The mutation rate of the human mtDNA deletion mtDNA4977.

The human mitochondrial mutation mtDNA4977 is a 4,977-bp deletion that originates between two 13-bp direct repeats. We grew 220 colonies of cells, each from a single human cell. For each colony, we counted the number of cells and amplified the DNA by PCR to test for the presence of a deletion. To estimate the mutation fate, we used a model that describes the relationship between the mutation rate and the probability that a colony of a given size will contain no mutants, taking into account such factors as possible mitochondrial turnover and mistyping due to PCR error. We estimate that the mutation rate for mtDNA4977 in cultured human cells is 5.95 x 10(-8) per mitochondrial genome replication. This method can be applied to specific chromosomal, as well as mitochondrial, mutations.     

The multiple ADP/ATP translocase genes are differentially expressed during human muscle development.

The expression of the genes encoding the three isoforms of the human ADP/ATP translocase (T1, T2, and T3) has been analyzed at different stages of myogenic differentiation in an in vitro muscle cell system and compared with that in mature muscle. The results indicate that the three stages of muscle differentiation corresponding to myoblast proliferation, myotube formation, and mature muscle fibers are characterized by a different pattern of expression of the ADP/ATP translocase genes. In particular, the two T2-specific mRNAs are present at high, similar levels in myoblasts and myotubes and markedly decrease in amount in mature adult muscle. By contrast, the T3-specific mRNA is present in high amount in growing myoblasts, decreases markedly in myotubes, and is barely detectable in adult muscle. Finally, the T1-specific mRNA is present at a high level in adult muscle and is not detectable in either myoblasts or myotubes. Therefore, T1 gene expression appears to be a marker of a late stage in myogenesis. A parallel investigation of expression of the myosin heavy chain mRNA revealed absence of hybridization with the specific probe in RNA from proliferating myoblasts, a significant hybridization in myotube RNA, and a strong signal in adult muscle RNA.     

The mtDNA-encoded ND6 subunit of mitochondrial NADH dehydrogenase is essential for the assembly of the membrane arm and the respiratory function of the enzyme.

Seven of the approximately 40 subunits of the mammalian respiratory NADH dehydrogenase (Complex I) are encoded in mitochondrial DNA (mtDNA). Their function is almost completely unknown. In this work, a novel selection scheme has led to the isolation of a mouse A9 cell derivative defective in NADH dehydrogenase activity. This cell line carries a near-homoplasmic frameshift mutation in the mtDNA gene for the ND6 subunit resulting in an almost complete absence of this polypeptide, while lacking any mutation in the other mtDNA-encoded subunits of the enzyme complex. Both the functional defect and the mutation were transferred with the mutant mitochondria into mtDNA-less (rho0) mouse LL/2-m21 cells, pointing to the pure mitochondrial genetic origin of the defect. A detailed biosynthetic and functional analysis of the original mutant and of the rho0 cell transformants revealed that the mutation causes a loss of assembly of the mtDNA-encoded subunits of the enzyme and, correspondingly, a reduction in malate/glutamate-dependent respiration in digitonin-permeabilized cells by approximately 90% and a decrease in NADH:Q1 oxidoreductase activity in mitochondrial extracts by approximately 99%. Furthermore, the ND6(-) cells, in contrast to the parental cells, completely fail to grow in a medium containing galactose instead of glucose, indicating a serious impairment in oxidative phosphorylation function. These observations provide the first evidence of the essential role of the ND6 subunit in the respiratory function of Complex I and give some insights into the pathogenic mechanism of the known disease-causing ND6 gene mutations.     

Alignment of the amino terminal amino acid sequence of human cytochrome c oxidase subunits I and II with the sequence of their putative mRNAs.

Thirteen of the first fifteen amino acids from the NH2-terminus of the primary sequence of human cytochrome c oxidase subunit I and eleven of the first twelve amino acids of subunit II have been identified by microsequencing procedures. These sequences have been compared with the recently determined 5'-end proximal sequences of the HeLa cell mitochondrial mRNAs and unambiguously aligned with two of them. This alignment has allowed the identification of the putative mRNA for subunit I, and has shown that the initiator codon for this subunit is only three nucleotides away from the 5'-end of its mRNA; furthermore, the results have substantiated the idea that the translation of human cytochrome c oxidase subunit II starts directly at the 5'-end of its putative mRNA, as had been previously inferred on the basis of the sequence homology of human mitochondrial DNA with the primary sequences of the bovine subunit.     

Injection of mitochondria into human cells leads to a rapid replacement of the endogenous mitochondrial DNA

Isolated human mitochondria containing a mitochondrial DNA (mtDNA) coded chloramphenicol resistance marker were injected into cells from two different human sensitive cell lines, 143BTK- and HT1080-6TG, which had been partially depleted of their mtDNA by ethidium bromide treatment. On the basis of the available evidence concerning the tolerance of introduced volumes into mammalian cells, it is estimated that, on the average, less than one mitochondrion was introduced into each cell. Under selective conditions, the mitochondria became established in the recipient cells with a frequency greater than 2-3 x 10(-3). An analysis of multiple mtDNA and nuclear DNA polymorphisms revealed a rapid replacement of the resident mtDNA by the exogenous mtDNA. Six to ten weeks after microinjection, this replacement was complete in all but one of the HT1080-6TG transformants, and nearly complete in the majority of the 143BTK- transformants. The quantitative behavior of the mtDNA of the transformants at very early stages of selection strongly suggests that intracellular mtDNA selection played a crucial role in this replacement, with significant implications for mitochondrial genetics.     

Human dihydrofolate reductase gene organization. Extensive conservation of the G + C-rich 5' non-coding sequence and strong intron size divergence from homologous mammalian genes

The complete human dihydrofolate reductase (DHFR) gene has been cloned from four recombinant lambda libraries constructed with the DNA from a methotrexate-resistant human cell line with amplified DHFR genes. The detailed organization of the gene has been determined by restriction mapping of the cloned fragments and DNA sequencing of all the protein coding regions and adjacent intron segments, and shown to correspond to that of the native human DHFR gene. The gene spans a length of approximately 29 X 10(3) bases from the ATG initiator codon to the end of the 3' untranslated region, and contains five introns that interrupt the protein coding sequence. The number and positions of introns are identical to those found in the mouse gene. By contrast, the size of the homologous introns (with the exception of the first one) varies greatly, up to several fold, in the genes from man, mouse and Chinese hamster; the intron sequences also exhibit a great divergence, except in the junction regions. A striking sequence homology, extending over several hundred nucleotides, exists between the human and mouse gene 5' non-coding regions. These regions are characterized by an unusually high G + C content, 72% and 66% in the human and mouse genes, respectively, which is maintained in the first coding segment and first intron, and is in sharp contrast to the relatively low G + C content (approximately 40%) of the remainder of the gene.