(Received for publication, February 25, 1997, and in revised form, April 1, 1997)
From the Institute of Biological Sciences, University
of Tsukuba, Ibaraki 305, Japan, the § Department of
Pathology, Saitama Cancer Center Research Institute, Saitama 362, Japan, the ¶ Institut Gustave Roussy, 39 rue Camille Desmoulins,
94805 Villejuif Cédex, France,
Rhone-Poulenc Rorer, Centre
de Recherche de Vitry-Alfortville 13, Quai Jules Guesde-BP14, 94403 Vitry Sur Seine, France, and the ** Department of Biochemistry,
Jichi Medical School, Tochigi 329-04, Japan
For isolation of mouse mtDNA-less
(0) cell lines, we searched for various
antimitochondrial drugs that were expected to decrease the mtDNA
content and found that treatment with ditercalinium, an antitumor
bis-intercalating agent, was extremely effective for completely
excluding mtDNA in all the mouse cell lines we tested. The resulting
0 mouse cells were successfully used for trapping the
mtDNA of living nerve cells into dividing cultured cells by fusion of
the
0 cells with mouse brain synaptosomes, which
represent synaptic endings isolated from nerve cells. With neuronal
mtDNA obtained, all of the cybrid clones restored mitochondrial
translation activity similarly regardless of whether the mtDNA was
derived from young or aged mice, thus at least suggesting that defects
in mitochondrial genomes are not involved in the age-associated
mitochondrial dysfunction observed in the brain of aged mice.
Furthermore, we could trap a very small amount of a common 5823-base
pair deletion mutant mtDNA (
mtDNA5823) that was
detectable by polymerase chain reaction in the cybrid clones. As the
amount of mutant mtDNA with large scale deletions was expected to
increase during prolonged cultivation of the cybrids, these cells
should be available for establishment of mice containing the deletion
mutant mtDNA.
Intercellular transfer of mtDNA between cultured mammalian cells has been used extensively for studying the contribution of mtDNA or cytoplasmic genetic factors to the expression of various phenotypes such as tumorigenicity (1, 2) and cell differentiation (3, 4). However, this mtDNA transfer system is problematic in that the mtDNA donor cells must be resistant to chloramphenicol for selective isolation of cells with exogenously transferred mtDNA (cybrids) (5, 6). Moreover, chloramphenicol selection and subsequent cultivation in the presence of chloramphenicol could not completely remove endogenous wild-type (chloramphenicol-sensitive) mtDNA in the host cells (7, 8). Therefore, the influence of the remaining host-cell mtDNA on the expression of the phenotypes cannot be excluded completely.
This problem could be overcome by using mtDNA-less (0)
cells as host cells. Recently,
0 cells were isolated
from avian (9) and human (10, 11) cells by treating the cells with
EtBr. Cytoplasmic transfer of mtDNA to these cells (particularly to
0 human cells) has been used extensively to provide
unambiguous evidence of whether accumulation of the candidate mutant
mtDNAs found in patients with mitochondrial diseases are responsible for the pathogenesis of the diseases (11-15), whether mtDNA is involved in the expression of tumorigenicity (16), and whether mitochondria fuse with one another and exchange contents (17, 18).
Furthermore, human 0 cells were used to study the
correlation between aging and mtDNA mutations. Our previous study
showed that the age-associated reductions in the activities of
mitochondrial translation and cytochrome c oxidase
(COX)1 (one of the mitochondrial
respiratory chain complexes) observed in fibroblasts from aged subjects
were not co-transferred to
0 HeLa cells together with
their mtDNA, suggesting that mtDNA mutations are not involved in at
least the age-associated mitochondrial dysfunction of human fibroblasts
(19). On the other hand, it has been reported that mitochondrial
dysfunction and accumulation of mtDNA mutations that have been shown to
be pathogenic in mitochondrial diseases are associated with aging,
particularly in human post-mitotic highly oxidative tissues such as
brain and muscles (20-23). Since fresh human tissues with highly
oxidative activities are difficult to obtain from healthy subjects,
mouse tissues must be used for further investigations of whether the
age-associated mitochondrial dysfunction in highly oxidative tissues is
due to the accumulation of somatic mtDNA mutations.
We reported previously that a common feature of age-associated changes
in both human and mouse mitochondrial respiratory functions is the
decrease in mitochondrial translational activity (24); therefore, mouse
brain can be used as a model to understand the causes of age-associated
mitochondrial dysfunction in non-dividing highly oxidative tissues.
Since mtDNA cannot be transferred from any mouse cells or tissues to
0 human cells due to the incompatibility between
mitochondrial and nuclear genomes of different species (25),
0 cell lines must be isolated from mouse cells for
further studies. However, exposure of mouse cells to EtBr (which has
been used effectively for isolation of
0 human cells)
frequently induced EtBr-resistant mutant cell lines containing mtDNA as
reported previously (26), and there have been no reports of successful
isolation of
0 cell lines from mouse cells.
In this study, we tested various chemicals that could be expected to
decrease the mtDNA content and finally succeeded in isolating 0 mouse cell lines. We then obtained cybrid clones with
neuronal mtDNA by fusion with brain synaptosomes (which are equivalent to synaptic endings isolated from nerve cells). These cybrids all
showed similar mitochondrial translation activity regardless of whether
the neuronal mtDNA was imported from young or aged mice, which at least
suggests that defects in mitochondrial genomes are not responsible for
the age-associated mitochondrial dysfunction observed in the brain of
aged mice. Furthermore, we trapped in the cybrid clones a very small
amount of a 5823-bp deletion mutant mtDNA (
mtDNA5823)
that was observed in mouse synaptosomes from all the individuals we
tested. No effective system has been established thus far for transfecting artificially mutagenized mammalian mtDNA into mitochondria for isolation of cells with pathogenic mutant mtDNA, but we could trap
mutant mtDNA accumulated in synaptosomes into
0 mouse
cells, and these should be available for isolation of mtDNA-knockout mice.
The mouse myoblast line C2C12 (C2 cells) and mouse fibroblast lines (5P cells derived from the B6mtJ strain and NIH3T3 cells) were grown in normal medium (RPMI 1640 (Nissui Seiyaku, Tokyo) containing 10% fetal calf serum, 50 µg/ml uridine, and 0.1 mg/ml pyruvate). A mouse pancreatic beta cell line (MIN6) was cultivated as described previously (27). The nuclear and mitochondrial genomes of all mouse cells except 5P cells are derived from Mus musculus domesticus. The mitochondrial genomes of 5P cells and the B6mtJ strain are derived from Mus musculus molossinus, whereas their nuclear genomes are from M. m. domesticus.
Isolation ofCells were plated at 1 × 102-5 × 104 cells/dish, and beginning 24 h after plating they were treated with ditercalinium (DC, an antitumor bis-intercalating agent) for 1 month. C2 and NIH3T3 cells were treated with 1.5 µg/ml DC, while MIN6 cells were treated with 56 ng/ml DC. The medium containing the drug was changed every 2 days. After 1 month, colonies growing in the medium were clonally isolated by the cylinder method. The cloned cells were then cultivated in normal medium without the drug.
Introduction of Synaptosomal mtDNA intoThe brain of a B6mtJ or B6 strain mouse was washed in
phosphate-buffered saline and homogenized in medium containing 0.25 M sucrose, 50 mM Hepes, pH 7.5, and 0.1 mM EDTA in a Teflon-glass Potter-Elvehjem homogenizer. The
homogenate was centrifuged at 1000 × g for 10 min at
4 °C, and the resultant supernatant was centrifuged at 17,000 × g for 20 min at 4 °C. The pellet was mixed with 5 × 106 0 C2 cells, and fusion was carried
out in the presence of 50% (w/v) polyethylene glycol 1500 (Boehringer
Mannheim). The fusion mixture was cultivated in selection medium (RPMI
1640 without pyruvate and uridine). On days 14-20 after fusion, the
cybrid clones growing in the selection medium were clonally isolated by
the cylinder method. Cybrid clones were cultivated in normal
medium.
Total cellular DNA (1-2
µg) extracted from 2 × 105 cells was digested with
the restriction enzyme (XhoI or BamHI (Nippon
Gene, Tokyo, Japan)), and restriction fragments were separated in 0.6% agarose gel, transferred to a nylon membrane, and hybridized with [-32P]dATP-labeled mouse mtDNA. The membrane was
washed and exposed to an imaging plate for 2 h, and
radioactivities of fragments were measured with a bioimaging analyzer
(Fujix BAS 2000, Fuji Photo Film, Tokyo, Japan).
For detection of a small amount of normal
mtDNA in 0 C2 cells, total cellular DNA (0.5 µg)
extracted from 2 × 105
0 C2
cells was used as a template. The nucleotide sequences from position
15495 to 15511 and from 15713 to 15697 were used as oligonucleotide primers. The cycle times were 60 s of denaturation at 94 °C,
60 s of annealing at 45 °C, and 120 s of extension at
72 °C for 30 cycles. The products were separated in 4% agarose gel.
For detection of large scale deletion mutant mtDNA in synaptosomes and
cybrids, PCR was carried out using 0.5 µg of total cellular DNA. The
nucleotide sequences from position 7558 to 7581 and from 13666 to 13642 were used as oligonucleotide primers. The cycle times were 30 s of denaturation at 94 °C, 30 s of annealing at 65 °C, and
120 s of extension at 72 °C for 30 cycles. This amplification
was repeated using an 0.02 volume of this mixture as a template. The
products were separated in 2% agarose gel. In these conditions, only
deleted mtDNA was amplified.
PCR products purified using a QIAEX II gel extraction kit (Qiagen, Hilden, Germany) were directly sequenced using a Taq polymerase kit (Prism) with fluorescent primers and dideoxynucleotides. Sequencing reactions were analyzed with an Applied Biosystems model 377 automatic sequencer.
Analysis of Mitochondrial Translation ProductsMitochondrial translation products were labeled with [35S]methionine as described previously (28) with slight modifications. Briefly, 2 × 106 cells in a culture dish were incubated in methionine-free medium containing 2% fetal calf serum for 45 min at 37 °C. The cells were then labeled with [35S]methionine for 2 h in the presence of emetine (0.2 mg/ml). Proteins in the mitochondrial fraction were separated by 0.85% SDS, 6 M urea, 12% polyacrylamide gel electrophoresis. For quantitative estimation of [35S]methionine-labeled polypeptides, the dried gel was exposed to an imaging plate for 12 h, and the radioactivities of polypeptides were measured with a bioimaging analyzer.
C2 cells of a mouse myoblast cell line were treated
with various antimitochondrial drugs, and the mtDNA contents of the
cells were then examined by Southern blot analysis. EtBr is effective for isolating 0 lines from avian (9) and human (10, 11)
cells, but we failed to isolate them from EtBr-treated mouse cells (the
copy number of mtDNA in C2 cells did not change substantially (Fig. 1A), whereas its transcription was completely
inhibited by EtBr treatment (data not shown), consistent with our
previous observations (26)). Similar results were obtained on treatment
of C2 cells with rhodamine 6G (29), dideoxycytidine (30), and
streptozotocin (31), even though these compounds were shown to be toxic
to mitochondria and were expected to decrease the mtDNA content (Fig. 1A). On the other hand, treatment with DC, an antitumor
bis-intercalating agent (32), was very effective for excluding mtDNA
not only in C2 cells but also in all the other mouse cell lines we
tested, such as NIH3T3 fibroblasts and a mouse pancreatic beta cell
line, MIN6 (Fig. 1B).
For isolation of 0 mouse cells, these mouse cell lines
were cultivated in the presence of DC for 1 month, and growing colonies were isolated clonally. Southern blot analysis showed no detectable mtDNA in any of these clones. We picked up one clone, named it
0 C2, and found that it did not recover mtDNA even after
3 months of cultivation in the absence of DC (Fig.
2A). Then, using the PCR technique, we
examined whether a small amount of mtDNA that was not detectable by
Southern blot analysis remained in the cells. Fig. 2B shows
that mtDNA was not amplified from a DNA sample prepared from
0 C2 cells. In these amplification conditions, mtDNA was
detectable when a DNA sample prepared from control parental C2 cells
was diluted as much as 1/104 (Fig. 2C). These
observations suggest that
0 C2 cells are entirely devoid
of mtDNA; thus, we had isolated
0 cells from the mouse
cell line. Moreover, these
0 C2 cells required pyruvate
and uridine in the medium for growth as do
0 avian (9)
and
0 human cells (10, 11).
The absence of mitochondrial translation activity in 0
C2 cells was confirmed by the absence of [35S]methionine
incorporation into mitochondrially synthesized polypeptides (Fig.
3B). Moreover, the activity of COX, a
mitochondrial respiratory chain complex, was completely lost in
0 C2 cells (Fig. 3C). These observations
suggest that complete depletion of mtDNA resulted in complete absence
of mitochondrial translation, leading to loss of the mitochondrial
enzyme activities involved in oxidative phosphorylation.
Characterization of
Before carrying out the transfer
of synaptosomal mtDNA from an aged mouse to 0 C2 cells,
it was necessary to confirm that the
0 C2 cells maintain
their abilities to receive and allow replication of exogenously
imported mouse mtDNA. Moreover, it was essential to exclude the
possibility that mtDNA-repopulated
0 C2 cells were not
revertant C2 cells containing recovered internal C2 mtDNA, which might
have remained in such a small amount that it could not be detected by
PCR (Fig. 2B). These possibilities were examined by fusion
of
0 C2 cells with synaptosomes prepared from the brain
of a B6mtJ congenic strain; this strain has the nuclear background of
M. m. domesticus but has mtDNA derived from a different
subspecies, M. m. molossinus, whereas C2 cells and most
mouse cells established from old inbred strains possess nuclear and
mitochondrial genomes derived from M. m. domesticus. Fig.
3A shows that M. m. molossinus mtDNA in 5P cells
derived from the B6mtJ strain and M. m. domesticus mtDNA in
C2 cells can be distinguished by their cleavages with the restriction
endonuclease BamHI.
Therefore, synaptosomes were prepared from a 6-week-old B6mtJ congenic
mouse, and after their fusion with 0 C2 cells in the
presence of polyethylene glycol, colonies growing in the selective
medium without uridine were isolated as cybrid clones Mol6-1, -2, and
-3 (Table I). No colonies were obtained from simple
mixtures of the
0 C2 cells and synaptosomes in the
absence of polyethylene glycol. Moreover, Southern blot analysis of
BamHI fragments unambiguously showed that all the cybrid
clones contained mtDNA of M. m. molossinus (Fig.
3A). If these clones were those of revertant C2 cells
obtained by the recovery of endogenous C2 mtDNA, they should show the
same restriction patterns as those of the parental C2 mtDNA, but this was not the case (Fig. 3A). Thus, the colonies growing in
the selective medium were cybrid clones containing imported
synaptosomal mtDNA. Restoration of mitochondrial translation activity,
indicated by [35S]methionine labeling of mitochondrially
synthesized polypeptides (Fig. 3B) and resultant restoration
of COX activity (Fig. 3C) were observed in the cybrid
clones. These observations suggest that
0 C2 cells have
the ability to receive synaptosomal mtDNA and allow its replication and
gene expression and that revertant clones with C2 mtDNA were not
isolated from
0 C2 cells.
|
The mitochondrial translation
activities in mouse synaptosomes increase progressively for 21 weeks
after birth and then gradually decrease with aging (24). Therefore,
mtDNAs from synaptosomes of 4-, 22-, and 75-week-old mice,
respectively, were introduced into 0 C2 cells by
polyethylene glycol fusion (Table I). The colonies growing in selective
medium without uridine were isolated as cybrid clones for determination
of the genomes that were responsible for the age-associated decline of
mitochondrial translation activity observed in synaptosomes. It is
unlikely that we preferentially selected only cybrid clones expressing
normal mitochondrial translation function, because cybrid clones with
very low mitochondrial translation activity can grow in the same
selective medium as used in this study (15).
Southern blot analysis of the XhoI restriction fragment
showed that the all cybrid clones contained mtDNA (Fig.
4A), suggesting that synaptosomal mtDNA of
non-dividing tissues could be recovered in dividing culture cell lines
by the use of 0 C2 cells. First, we compared the
mitochondrial translation activities of cybrid clones with imported
mtDNA from synaptosomes of young and aged mice by analyzing
[35S]methionine incorporation into mitochondrially
synthesized polypeptides. Fig. 4B shows that the amounts of
newly synthesized polypeptides encoded by mtDNA were similar when
synaptosomal mtDNAs from mice of different ages were introduced into
0 C2 cells. Therefore, the abnormalities of
mitochondrial respiratory function in the brain of aged mice (24) are
not co-transferred with the synaptosomal mtDNA to
0 C2
cells. This suggests that mtDNA is not involved in the observed age-related dysfunction of mouse brain mitochondria.
Next, we searched for large scale deletion mutations of mtDNA in
synaptosomes and in the cybrid clones using PCR techniques. All samples
of synaptosomes and some cybrid clones showed a fragment of about 1 kilobase, regardless of whether they were prepared from young or aged
mice, whereas this fragment was not observed in parental
0 C2 cells or C2 cells (Fig.
5A). These observations suggest the presence
of a common large scale deletion mutation in mouse synaptosomal mtDNA
that can be transferred to
0 C2 cells, although its
amount was not sufficient to be detected by Southern blot analysis
(Fig. 4A). Sequence analysis of the amplified fragment from
the cybrid clone Dom75-4 showed that the common deletion was 5823 bp
long with a break point from nucleotide positions 7819 within the
ATP8 gene to 13641 within the ND6 gene and that
the deletion was flanked by a 5-bp direct repeat (TACCC) (Fig.
5B). Therefore, we can trap the mouse somatic mutant mtDNA with a common 5823-bp deletion (
mtDNA5823) into
the cybrid clones, and its amount can be expected to increase during
prolonged cultivation.
There are no previous reports on successful isolation of
0 lines from mouse cells, probably because exposure of
mouse cells to EtBr, which has been effectively used for isolation of
0 lines from yeast, avian, and human cells (33), induced
EtBr-resistant mutant cell lines containing mtDNA instead of isolating
0 lines. In this study, by screening drugs that were
expected to decrease the mtDNA content, we found that only DC is
effective for elimination of mouse mtDNA and isolation of
0 lines from various mouse cell lines.
Mouse 0 cells isolated from myoblast C2 cells
(
0 C2 cells) were effective for investigation of the
correlation between age-associated mitochondrial dysfunction and
accumulation of somatic mtDNA mutations in highly oxidative tissues.
Mouse
0 cells are necessary for study of this problem,
since human
0 cells cannot be used for the following two
reasons. One is that fresh human tissues with highly oxidative
activities are very difficult to obtain from healthy subjects. The
other is that
0 human cells do not accept mouse mtDNA
from any tissues due to incompatibility between mitochondrial and
nuclear genomes of different species (25). In this study, neuronal
mtDNA was transferred to
0 C2 cells by their fusion with
synaptosomes prepared from the brains of young and aged mice. We
obtained several mtDNA-repopulated cybrid clones and examined whether
the reduced mitochondrial translation property observed in brain
mitochondria of aged mice was co-transferred with mtDNA. Results showed
that all cybrid clones with imported mtDNA from either young or aged
mice had similar mitochondrial translation activity (Fig.
4B), suggesting that defects in the mitochondrial genome,
even if they are present and have progressed with aging, are not
responsible for the age-associated mitochondrial dysfunction observed
in synaptosomes of aged mice.
It is unlikely that we selected cybrids with normal mitochondrial
translation preferentially, because human cybrid clones with very low
mitochondrial translation activities due to the predominance of
pathogenic mtDNA mutations have been shown to grow in the same
selective medium as that used in this study (15). Although our
synaptosomal fraction might be contaminated with glial mitochondria,
the possibility that we introduced glial mtDNA into 0 C2
cells was also excluded by the fact that mitochondrial transfer was
limited solely to the situation when they were surrounded by an intact
cell membrane, as with mitochondria in synaptosomes or in cytoplasts,
and mitochondria alone were not imported into cells by the cell fusion
techniques. It was also unlikely that nuclear genomes of non-enucleated
neuronal cells were introduced into the cybrid clones because the modal
chromosome number of the cybrid clones (71) was comparable to that of
the recipient
0 C2 cells.
It has been generally thought that somatic mutations are more likely to
accumulate in mtDNA than in nuclear DNA because mtDNA is a target of
most mutagens and is always exposed to oxygen-free radicals produced in
mitochondria but is not protected by proteins like histones (34).
Moreover, accumulation of various somatic mutations in mtDNA and the
resultant decline of mitochondrial respiratory function during a
lifetime has been proposed to be involved in aging processes. For
example, mitochondrial respiratory functions were reported to decrease
with aging in highly oxidative tissues, and these processes
appeared to be associated with accumulation of pathogenic mtDNA
mutations during aging (35-37). However, it was possible that the
age-associated accumulation of mtDNA mutations are not necessarily
responsible for age-associated mitochondrial dysfunction, since
mitochondrial respiratory functions are controlled by both
mitochondrial and nuclear genomes and the nuclear genome encodes most
mitochondrial proteins including factors necessary for replication and
expression of the mitochondrial genome (33, 38, 39). Our study using
0 C2 cells solved this problem, showing that defects in
the mitochondrial genome, if present, are not involved in
age-associated mitochondrial dysfunction.
These observations are consistent with our previous observations that
the phenotypes of reduced activities of COX and mitochondrial translation observed in fibroblasts from aged subjects were not co-transferred to 0 HeLa cells together with their mtDNA
(19). Therefore, the conclusions from results on human fibroblasts
could be extended to non-dividing, highly oxidative tissues.
In this study, we also found that mtDNA5823, commonly
observed in mouse synaptosomes, was introduced into some cybrid clones. The trapping of mtDNA with a large scale deletion mutation in synaptosomes into dividing cultured cells will enable us to produce mtDNA-knockout mice. Even though the amount of the
mtDNA5823 in the cybrid clones was very small, it would
proliferate faster than the wild-type mtDNA (40) and eventually
accumulate during prolonged cultivation as does human deletion mutant
mtDNA in the cybrids (11). We are now trying to establish
mtDNA-knockout mice using the cybrid clone with
mtDNA5823; when mitochondria containing mouse
mtDNA5823 are introduced into fertilized mouse eggs by
microinjection, mtDNA-knockout mice should be obtained. These
should be useful as models of human mitochondrial diseases, aging,
diabetes, and age-related neurological diseases and for studies on the
mechanisms of transmission of mutant mtDNA and expression of its
pathogenic effects in various tissues.
Mouse 0 lines were also available for studying the
functional consequences of mtDNA depletion in differentiated cells. For example, a
0 line isolated from the mouse pancreatic
beta cell line MIN6 was used for studying the influence of mtDNA
depletion and its repopulation on phenotypic expression of
glucose-stimulated insulin secretion (27).