THEJOURNAL

OF BIOLCGICAL
CHEMISTRY Vol. 269, No. 9, Issue of March 4, pp. 6878-6883, 1994
0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U S A

Nuclear but Not Mitochondrial Genome Involvement in Human

Age-related Mitochondrial Dysfunction
FUNCTIONAL INTEGRITY OF MITOCHONDRIAL DNA FROM AGED SUBJECTS*

(Received forpublication, May 19, 1993, and in revised form, November 9, 1993)

Jun-Ichi Hayashi$$$ Shigeo Ohtall, Yasuo Kagawall, Hiroshi Kondo**, Hideki KanedaSS,
Hiromichi YonekawaSS, Daisaku TakaiS, and Shigeaki Miyabayashigl
From the $Institute of Biological Sciences, University of Tbukuba, Tbukuba Ibaraki 305, Japan, $Department of
Biochemistry, Saitama Cancer Center Research Institute, Saitama 362, Japan, lpepartment of Biochemistry, Jichi
Medical School, Zbchigi 329-04, Japan, **Department of Biology, lbkyo Metropolitan Institute of Gerontology, lbkyo 103,
Japan, $$Department of Laboratory Animal Science, lbkyo Metropolitan Institute of Medical Science, Zbkyo 113, Japan,
and $§Department of Pediatrics, Tohoku University School of Medicine, Sendai 980, Japan

The role of mtDNA and nuclear genomein human ag- mtDNA transcription in rat brain (12) and translation in rat
ing was examined by their intercellular transfer using skeletal muscle (13) are also reported to decrease withage.
skin fibroblastsandmtDNA-lessHeLa cells (pO-HeLa However, there is as yet no convincing evidence that accu-
cells). We found in vivo age-related reductions in the mulated mtDNA mutations are causal genetic factors of age-
activity of cytochrome c oxidase in human skin fibro- related mitochondrial dysfunction. Moreover, this age-related
blastsobtainedfrom 16 donorsofvariousages (0-97 phenotypic change may beunder control of the nucleargenome,
years). The abnormality in mitochondria of the aged do- because nuclear genes encode most mitochondrial proteins in-
nors was not attributable to either decrease in the copy cluding all the factors involved in expression of the mitochon-
numberofmtDNAmolecules or increase in the copy drial genome (14,151. This problem can be solved by examining
number ofdeletion mutantmtDNA molecules, but to sig- whether mtDNA in cells of aged humans and age-related mi-
nificant decrease in overall polypeptide synthesis in the
tochondrial dysfunction are co-transferred to othercells. Such
mitochondria. However, intercellular mtDNA transfer
intercellular mtDNA transfer is technically possible when cul-
experiments showed that fibroblast mtDNA from elderly
donors is functionally intact. By contrast, intercellular tured cells from aged donors are available.
transfer of HeLanuclei to fibroblasts from aged donors Since the discovery of the intrinsic limitof cell division, i.e.
restored cytochrome c oxidase activity, suggesting that the limit of population doublings of human diploid fibroblasts
the age-related phenotype was nuclear recessive. How-grown in culture(161, these cells have been used extensivelyas
ever, during subsequent cultivation of these hybrids, a model
the system for studying in vitro cellular aging (17). Gold-
activity gradually reduced again, associated with stein et al.(18)explored the relationshipbetween their limited
gradual chromosome loss. These observations support replicative life span and energy metabolism, using fibroblasts
the idea that accumulation of nuclear recessive somatic from the samedonor at early and late passages, and found that
mutations, but not mtDNA mutations, is responsible for there was no gross deficit in energy metabolism at increased
the in vivo age-related mitochondrial dysfunction ob- population doubling levels (PDL), i.e. during i n vitro aging of
served in humanskin fibroblasts. cultured human fibroblasts.
On the other hand, in the present study we observed in vivo
age-related reduction of COX activity in cultured human skin
Aging is a complex biological phenomenon associated with fibroblasts from 16 different donors of variousages (0-97
time-related degenerativeprocesses. Recently, accumulation of years). The abnormality in mitochondria of the aged humans
various somatic mutations inmtDNA during a lifetime and the was not attributable to either decrease in thecopy number of
resultant decline of mitochondrial energy production have been mtDNA molecules or increase in the copy number of deletion
proposed to be involved in aging processes (1-3) and in several mutant mtDNA molecules, but toa significant decrease inover-
degenerative diseases (1,3). In fact, there are reports that the all polypeptide synthesisinthe mitochondria. Intercellular
number of cytochrome c oxidase(COX)l-negativefibersin- transfer of mtDNA and nuclear genome using pO-HeLa cells,
creases with age in human muscle (4, 5), and that mitochon- which completely lack mtDNA (19, 20), showed that nuclear
drial respiratory function decreases with age in humanmuscle genome, but not mtDNA, of elderly donors was responsible for
(6)and liver(7). This age-related decrease in energy production the in vivo age-related down-regulation of mitochondrial en-
is proposed to be caused by the accumulation of a common ergy production found in human skinfibroblasts.
mutant mtDNA with a 4977-base pair deletion (AmtDNA4977)
in human brain (81,heart (9, lo), and liver (11).The rates of EXPERIMENTAL PROCEDURES
Cells and Cell Culture-Normal human skin fibroblasts lines derived
from 16 different donors of various ages (0-97 years) were obtained
* This work was partly supported by Grants-in-aid for ScientificRe- from the Tokyo Metropolitan Institute of Gerontology,Japanese Cancer
search from the Ministry of Education,Science and Culture, Japan. The Research Resources Bank, and Tohoku University School of Medicine
costs of publication of this article were defrayed in part by the payment (see Fig. 1). po-HeLa cells, which are resistant to 20 p~ 6-thioguanine,
of page charges. This article must thereforebe hereby marked "adver-
tisement" in accordance with 18 U.S.C. Section 1734 solely to indicate and fibroblast lines were grown in glucose-rich medium RPMI 1640
this fact. supplemented with pyruvate (0.1 mg/ml) and 10% fetal bovine serum
1 To whom correspondence should be addressed. "el.: 298-53-6650; (20).
Fax: 298-53-6614. Intercellular Dansfer of Fibroblast mtDNA-Intercellular transfer of
The abbreviations use are: COX, cytochrome c oxidase; PDL, popu- mtDNA wa8 carried out as described previously(20)by fusion of enucle-
lation doubling level; PCR, polymerase chain reaction. ated fibroblastswith po-HeLa cells and cybrid clones were isolated in

6878
 Functional
Integrity of Human
mtLNA
from
Aged
Subjects 6879
selective medium. Briefly, skin fibroblasts grown on round glass disks b
were enucleated by centrifugation (23,000 x g, at 34 "C for 10 min) in
the presence of cytochalasin B (Sigma; 10 pg/ml). The resulting cyto-
plasts were mixedwith po-HeLa cells,and fusion was camed out in the
presence of 50% (wlv) polyethyleneglycol 1500 (BoehringerMannheim).
The fusion mixture was cultivated in selective medium without glucose
(DM170, Kyokuto Kagaku, Tokyo) supplemented with 20 6-thiogua-
nine and 10% fetal bovine serum (20). The residual non-nucleated pa-
rental fibroblasts and hybrids between non-nucleated fibroblasts and
po-HeLa cells were completely eliminated with 20 p~ 6-thioguanine.
Unfused parental po-HeLa cells were removed by culture in DM170
medium, since they could not grow in the medium without glucose due
to the complete absence of mtDNA (20). On day 14 after fusion, cybrid
colonies grownin selective medium were harvested and cloned by the
cylinder method (21).Cybrids were cultivated in normal medium (RPMI
1640 + pyruvate + 10% fetal bovine serum). As a control, HeLa mtDNA
was introduced into po-HeLa cells by the fusion of po-HeLa cells with
enucleated wild-type HeLa cells, which are sensitive to 6 - ~ 0 ~ ~ n e .
The cybrids named HeEB cells, i.e.pO-HeLa cells containing mtDNA
from wild-type HeLa cells, were isolated in selective medium DM170
with 20 p &thioguanine, in which unfused pO-HeLa cells, wild-type
HeLa cells, and their hybrids could not survive. Donor age (years) PDC
Intercellular Dansfer of H e h Nuclear Genome-Intercellular trans-
fer of HeLa nucleito TIG102 fibroblasts from a 97-year-old womanwas FIG.1. Biochemical analysis of COX activities of 16 lines of
attained by fusion of TIGlO2 fibroblasts with po-HeLa cells using pol- cultured human skin fibroblasts isolated from donors ofvarious
yethylene glycol 1500 (21). The fusion mixture was selected with ages. a , influence of in vivo age on COX activity. Donor age, sex, and
hypoxanthindaminopteridthymidine medium (Sigma), and colonies PDL, i.e. in vitro aging levels, of fibroblasts are indicated in parenthe-
ses. 1 , TIG3S (fetus, M, 19 PDL); 2 , TUMP403 (1year, M, 8 PDL); 3 ,
grown in the selection medium were cloned as described above. TUMP304 (10 years, F, 9 PDL); 4, TUMP415 (20 years, F, 8 PDL); 5,
Chromosome Analysis-The chromosome compositionsof cybrid and TUMP300 (27 years, M, 8 PDL); 6, TUMP372 (30 years, M, 7 PDL); 7,
the hybrid clones of log-phase cells were analyzed immediately after TIG114 (36 years, M, 18 PDL); 8, TUMP391 (37 years, M, 7 PDL); 9,
cloning using air-dried chromosome preparations as described previ- TUMP474 (40 years, F, 6 PDL); 10, TIG112 (40 years, F, 14 PDL); 11,
ously (22). At least 20 metaphase spreads of each clone isolated were TIG108 (40years, F, 15 PDL); 12, TUMP386 (57 years, M, 8 PDL); 13,
counted. TUMP363 (65 years, F, 7 PDL); 14, TIG103 (69 years, F, 14 PDL); 15,
Southern Blot Analysis-Total DNA (5 pg) extracted from 2 x lo5 TIG106 (80 years, F, 13PDL); 16, TIGlO2 (97 years, F, 16 PDL).All TIG
cells was digested with the single-cut restriction enzyme PvuII to esti- fibroblast lines except TIG103, TIG106, and TIGlO2 wereobtained fmm
mate the contents of mtDNA in fibroblasts from fetus and elderly do- the Tokyo Metropolitan Institute of Gerontology. TIG103, TIGlO6,and
nors, or digested with HhaI to determine whether mtDNAof the cybrids TIGlO2 were obtained from the Japanese Cancer Research Resources
was derived from mtDNAofthe donor cells.The fragments separated by Bank. All TUMPfibroblast lines were obtained from the Department of
Pediatrics, Tohoku University School of Medicine.6 , influence of in vitro
agarose gel electrophoresis were then transferred to nitrocellulose age on COXactivity. 6, TUMP372 fibroblasts; 12, TUMP386 fibroblasts.
membranes and hybridized with [~-3*PldCTP-labeledHeLa mtDNA.
The membranes were washed and exposed to x-ray film for 1 h a t
-80 "C. To quantitate the contents ofmtDNAof normal size in the
fibroblasts, the membranes were exposed to imaging plates (Fuji Film, ~ E S ~ ~
Tokyo) for 5 min, and radioactivity was measured with a bioimage COX Activities of Normal Skin F ~ b ~ b l a s t s O b from ta~~d
analyzer, Fujix BAS 2000 (Fuji Film). Donors of Various Ages-In this study, 16 lines of cultured
PCR Analysis-Total DNA (100 ng/ml) extracted from 2 x lo6 cells normal human skin fibroblasts isolated from 16 persons of vari-
was used for amplification.The nucleotide sequence from position7901
ous ages (0-97 years) were used for studying "in vivo aging."
to 7920 on the light strand and from 13650 to 13631 on the heavy
strand, which have frequently been used ( 8 , l l ) for detection of a com- First, we compared the COX activities of these fibroblasts and
monA-mtDNA4977,wereused as oligonucleotide primers. The cycle found that the activities decreased gradually as the fibroblast
times were 15-s denaturation at 95 "C, 15-s annealing at 50 "C, and donors' age increased (Fig. la). Although the PDL, i.e. in vitro
120-9 extension at 72 "C for 30 cycles. The products were separated on aging levels, of cultured human fibroblasts lines examined
agarose gels containing ethidium bromide (0.5 pg/ml). were not constant, they did not affect the COX activities (Fig.
Analysis of Mitochondrial Panslation Products-Mitochondrial l b ) , consistent with reports by Goldstein et al. (18)and Sun et
translation products were labeled with Q5S]methionineas described
al. (26). These results suggest that fibroblast lines from donors
elsewhere (23) with slight modifications. Briefly, semiconfluent cells in
a dish were incubated in methionine-free medium containing 10%fetal of various ages provide a good model system for studying in
bovine serum for 1h at 37 "C.Then, the cells werelabeled with [35Slme- vivo aging.
thionine for 1h in the presence of emetine (0.15 mg/ml). The mitochon- The above data represent overall COX activities of the cells,
drial fraction was obtained by homogenization in 0.25 M sucrose, 1m~ not the activities of single cells. To determine whether a small
EGTA, 10 m Hepes-NaOH, pH 7.4, followed by differential centrifu- number of fibroblasts, such as from muscle fibers of elderly
gation. Proteins in the mitochondrial fraction (50 pgflane) were sepa- donors (51, show normal COX activities, we carried out a cyto-
rated by SDS-urea-polyacrylamidegel electrophoresis. The dried gel chemical analysis of COX activities at theindividual cell level
was exposed to an imaging plate for 6 h, and the labeled polypeptides
were located with a bioimaging analyzer. mtDNA contents were meas- using TIG102 fibroblasts from a 97-year-old womanand TIG3S
ured by dot blot hybridization. The ~ t o c h o n d n awere lysed in 0.5 M fibroblasts from a fetus. A s shown in Fig. 2, the COX activities
NaOH, 1.5 M NaCl and blotted directly onto a nylon membrane at serial in TIGlO2 fibroblasts were much lower than those in TIG3S
dilutions. Purified human mtDNA was used as a standard. Incorpora- fibroblasts, and thedecrease was uniform in all the individual
tion of ~ ~ ~ S l m e ~ ointo
n i nthe
e total protein fraction was measured cells (Fig. 26).
with a liquid scintillation counter. Comparison of Total Amounts of mtDNA and~ ~ t o c h o n d r i a ~
Analysis of COX Activity-For biochemical analysis, log-phase cells
Banslation Products in Aged and Fetal Human Fibroblasts-
were harvested, and COX activity was measured as the rate of cyanide-
sensitive oxidation of reduced cytochromec as previously described (24). Since three subunits of COX are encoded by mtDNA, the ob-
For cytochemical analysis, cells grown on coverslips were fixed with served age-related reduction of COX activity could be due to
freshly prepared 4% glutaraldehyde, 0.1 M phosphate buffer, pH 7.4, for reduction of the amount of normal mtDNA or to down-regula-
10 min, and stained with COX (25) for 2 h at 37 "C. tion of post-transcriptional activity in themitochondria. To ex-
6880 Functional Integrity of Human
mtDNA
from Aged Subjects
was detected in DNA samples of TIGlO2 fibroblasts from the
g e d donor or fetal TIG3S fibroblast, evenunder theconditions
of PCR amplification that could detect 0.002% of AmtDNA4977
molecules in control samples (Fig. 3b).
Second, to examine whether mitochondria of aged subjects
show down-regulation of post-transcription, we compared the
activities for protein synthesis of mitochondria in fibroblasts of
the aged subject and the fetus. Mitochondrial translation prod-
ucts were analyzed by SDS-polyacrylamide gel electrophoresis
after pulse-labeling the fibroblasts with [35S]methionine in the
presence of emetine, a specific inhibitor of protein synthesis in
the cytoplasm. In TIGlO2 fibroblasts, significant decreases of
radioactivity were observed not only in three COX subunits
(COI, COII, and COIII) but also in all other polypeptides en-
coded by mtDNA (Fig. 4). These results suggest that reduc- the
tion in[35S]methionine label incorporated into fibroblasts from
aged subjects is the result of a decrease in overall mitochon-
drial protein synthesis, or the resultof an increase in the turn-
over of mitochondrial translation products due to the lack of
nuclear-encoded gene productswith which to assemble into the
inner membrane protein complexes.
Intercellular Dansfersof Fibroblast mtDNA from Aged Sub-
ject and Fetus to Po-HeLu Cells-Since subunits ofCOX are
encoded by nuclear and mitochondrial genomes and since mi-
tochondrial protein synthesis is under the control of both ge-
nomes, it was necessary to determine which genome was re-
sponsible for the age-related down-regulation of mitochondrial
protein synthesis and COX activity. For this purpose we used
the fetalfibroblast line TIG3S and fibroblast line TIGlO2 from
a n old subject as mtDNA donors. pO-HeLa cells, which have no
mtDNA and no COX activity (20), were usedas mtDNA recipi-
ent cells. On transfer of mtDNA from these two types of fibro-
blasts to po-HeLa cells, several cybrid clones were obtained,
respectively. As a control experiment, we introduced HeLa cell
mtDNA into po-HeLa cells and isolated cybrids (named HeEB)
with HeLa mtDNA. Karyotype analysis showed that all the
cybrid clones had a modal chromosome number of 50 (range,
47-54), which was the same as that of pO-HeLa cells (50; range
47-52). On the other hand, the restriction patterns with HhaI,

- .. .
, ' D
which can distinguishHeLa mtDNAfromother humanmtDNA
(27, 281, showed that themtDNA in all the cybrid clones were
derived exclusively from those of the mtDNA donor cells.
FIG.2. Cytochemical analysis of COX activities of skin fibro- Comparison of Mitochondrial Functions of Cybrid Clones
blasts of a fetusand an old person. Cells were stained cytochemi- with mtDNA of Fibroblasts from Aged and FetalDonors-The
cally for COX activity. a, TIGBS fibroblasts isolated from a fetus; b,
TIGlO2 fibroblasts isolated from a 97-year-old woman. Bar, 80 pm. COX activities and mitochondrial protein syntheses of cybrid
clones with mtDNA from TIG3S and TIGlO2 fibroblasts were
compared. As shown in Figs. 4 and 5, irrespective of whether
amine thesepossibilities, we first compared the total amounts the mtDNApopulation was imported from fibroblasts of aged or
of mtDNA per cell in TIGlO2 and TIG3S fibroblasts by South- fetal donors, all the cybrid clones showed similar activities of
ern blot analysis after digestion of total DNA samples with a COX and mitochondrial protein synthesis to those of cybrid
single-cut restriction enzyme, PuuII (Fig. 3a). Theresults clone HeEB with HeLa mtDNA. Since COX activity was re-
showed that there were no large mtDNA deletions in the duced similarlyin individual TIGlO2 fibroblasts (Fig. 2b),
mtDNA of either the aged subject or the fetus, and that the these cybrid clones were unlikely to be restricted to those with
amount of normal sized mtDNA was slightlyincreased in mtDNA imported from TIGlO2 fibroblasts with normal COX
TIGlO2 fibroblasts from the aged donor, consistent withprevi- activity. Accordingly, the phenotype of reduced mitochondrial
ous observations (27,28).Thus theage-related decrease of COX function observed in fibroblasts of the aged subject was not
activity in fibroblasts (Figs. 1and 2) is not due to a decrease in co-transferred to pO-HeLa cells with the mtDNA. Similar re-
the copy number of mtDNA or to the accumulation of large sults were obtained when mtDNA of fibroblast line TIGlO6
scale deletion mutations in themtDNA. from another aged subject was introduced into po-HeLa cells.
Recently, mtDNA molecules with large scale deletion muta- Moreover, on PCR analysis, no AmtDNA4977molecules were
tions that were not detectable by Southern blot analysis were found in any cybrid clone (Fig. 3b). As all the cybrid clones
observed in human brain,muscle, and liver by the PCR tech- share the same nuclear background as HeLa cells, these obser-
nique. Of the largescale deletions, a 4977-base pair deletion of vations clearly suggest that mtDNA molecules in fibroblasts
mtDNA was found to accumulate in these organs with increase from the aged subject are functionally intact. Therefore, the
in age(3).Using the PCR amplification conditionsdescribed by age-related mitochondrial dysfunction observed in fibroblasts
Ikebe et al. (81, we examined whether the AmtDNA4977was of old subjects is not due at least to the accumulation of various
present in aged or fetal fibroblasts. However, no AmtDNA4977 kinds of somatic mutations inmtDNA.
 Functional Integrity of Human
mtDNA from Aged Subjects 6881
a b

@Ft Ag
.Experimental
Control
~ 1 2 3 4 5 1 2 3 4 5 6 7 " w

16 kbp- 5~~~~~~~ 1'612

7 0 bp

FIG.3. Southern blot and PCR analyses of mtDNka , comparison of amounts of mtDNA in TIG3S and TIGlO2 fibroblasts by Southern blot
analysis of PuuII restriction patternsof mtDNA. po. po-HeLa cells; Ft, TIG3S fibroblasts from a fetus; Ag, TIGlO2 fibroblasts from a 97-year-old
woman. Total DNA (5 pg/lane) extracted from the cells was analyzed. b, screening of A-mtDNA4977in fibroblasts and cybrid clones by PCR
amplification. Thecontrol DNA sample containing20% A-mtDNA4977prepared from a patient with Kearns-Saye syndrome (kindly provided from
Drs. R. Sakuta and I. Nonaka, National Centerof Neurology and Psychiatry, Japan) was serially diluted with total DNA prepared from TIG3S
fibroblasts to determinethe minimal detectable contentof A-mtDNA4977in our PCR amplification conditions (see "Experimental Procedures"). PO,
p"-HeLa cells; Control 1 , 2 , 3 , 4 , and 5 are DNA samples containing 2, 0.2, 0.02, 0.002, and 0.0002% A-mtDNA4977,respectively; Experimental 1,
2 , 3 , 4 , 5 , 6 ,and 7 are DNA samples of TIG3S and TIG102 and cybrid clones Ft2, Agl, Ag2, Ag3, and HeEB,respectively. Ft2 isa cybrid clone with
TIG3S mtDNA; Agl, Ag2, and Ag3 are cybrid clones with TIG102 mtDNA HeEB is a cybrid clone with HeLa mtDNA. M w , molecular weight
standards (+X174/HincII digests; Toyobo, Osaka). Fragmentsof 5750 and 773 base pairs are PCR products amplified from wild-type mtDNA and
from A-mtDNA4977,respectively.
b
I
1 2 3 4 5 6 7 8 9 3

-NO5
-coI
- NO4
-Qtb
-NO2
-NO1
-COll
- co Ill
-ATP6

-NO3
-ATP8
-ND4L 1 2
FIG.5. Comparison ofactivities inCOX (a)and mitochondrial
translation ( b )of cells with mtDNA derived fromfetal and aged
human skin fibroblasts. U, cells with TIG3S fibroblast mtDNA; m,
FIG.4. Analysis of mitochondrial proteinsynthesis in skinfi- cells with TIGlO2 fibroblast mtDNA; Ed, cells with HeLa mtDNA. I ,
broblasts from a human fetus, an aged human subject, and cy- TIG3S fibroblasts;2, TIGlO2 fibroblasts; 3 and 4 , cybrid clones Ftl and
brids. 1, TIG3S fibroblasts; 2, TIGlO2 fibroblasts; 3, pn-HeLa cells; 4 , Ft2 (Po-HeLa cells imported mtDNA from TIG3S fibroblasts);5 , 6, and
cybrid clone Ft2 (pO-HeLa cells imported mtDNA from TIG3S fibro- 7, cybrid clones Agl, Ag2, andAg3 (p"-HeLa cells imported mtDNAfrom
blasts); 5,6, and 7 are cybrid clones Agl, Ag2, and Ag3 (pO-HeLa cells TIGlO2 fibroblasts); 8, cybrid clone HeEB (pO-HeLa cells imported
imported mtDNAfrom TIGlO2 fibroblasts), respectively; 8,cybrid clone mtDNA from HeLacells).
HeEB (p"-HeLa cells imported mtDNA from HeLa cells); 9, HeLa cells.
ARer specific [35Slmethioninelabeling of mitochondrialtranslation
products in the presence of emetine, proteinsof the mitochondrial frac- amined their COX activity. The results showed that the re-
tion (50 pg/lane) were separated by SDS-urea-polyacrylamide gelelec-
trophoresis. ND5, COI, ND4, Cytb, ND2, NDl, COII, COIII, ATP6, ND3, duced COX activity in the agedfibroblasts recovered com-
ATP8, and ND4L are polypeptides assigned to mtDNA genes. pletely with the introduction of HeLa nuclei (Fig. 6 ) ,suggesting
that
human age-related
mitochondrial dysfunction was
Intercellular Dansfer of HeLa Nuclei to Fibroblasts of Aged nuclear-recessive. Since HeLa nuclei-donor cells, i.e. po-HeLa
Subject-Then, to examine whether thereduced COX activity cells, showed no COX activity (Fig. 6), restoration of COX ac-
in cultured fibroblasts is inherited in a nuclear-dominant way tivity in thehybrids must be the resultof cooperation between
or a nuclear-recessive way, HeLa nuclei were introduced to the imported HeLa nuclear genome and mtDNA of host fibro-
TIGlO2 fibroblasts. Since pO-HeLa cells were completely with- blasts of the aged subject.
out mtDNA, introduction of HeLa nuclei to the aged fibroblasts During their subsequent cultivation, however, the hybrids
was attained simply by the fusion of TIGlO2 fibroblasts with gradually lost COX activity (Fig. 6 ) in a similar way to the
pO-HeLa cells. We subsequently isolated their hybrid clones in gradual age-related loss ofCOX activity observed in human
the selective medium (cf. "Experimental Procedures") and ex- skin fibroblasts (Fig. 1).Moreover, the gradual loss ofCOX
6882 Functional Integrity of Human mtDNA from Aged Subjects
presumably do not accumulate in dividing tissues due to selec-
tion against the survival of cells containing these deletion mu-
tants (291, but theycould be propagated predominantly in blood
cells of cases of Pearson syndrome (35) and in cybrid clones
isolated by fusion of po-HeLa cells with enucleated fibroblasts
from a patient with Keams-Sayre syndrome (20).Accordingly,
it is also unlikely that deletion'mu~tionsin mtDNA of fibro-
blasts from aged subjects were selectively eliminated during

1
the enucleation, cell fusion, and cloning processes. These con-
siderations indicate that accumulation of various kinds of
mtDNA somatic mutations, even if it occurs as supposed in
aged brain andmuscle, is not involved in thedonor age-related
decrease in mitochondrial respiratory function observed in hu-
man skin fibroblasts (Fig. la).
The accumulation of somatic mutations in mtDNA is sup-
n posed to play a significant role in carcinogenesis (36-39) and
aging (1-3) for the following reasons. Mitochondria are highly
- 0
b
:"
z
po -1 -2 -3 oxygenic organelles due to their energy production function;
Parents Hybrids
mtDNA lacks histones, which protect DNA from mutagenic
damage; and mtDNA repair systems are limited. Moreover, this
FIG.6. Recovery of the reduced COX activity in the aged fibro-
blasts by introduction of HeLa nuclei. po, po-HeLa cells; TIG10.2, possibility is supported by recent findings that the hydroxyl
TIG102 fibroblasts from a 37-year-old woman; Hybrids, TIGlO2 fibro- radical adduct, 8-OH-dG (40), very small amounts of deletion
blasts with imported HeLa nuclei. Hybrids-1, -2, and -3 are COX ac- mutations (8-11,31,32,41,421, and a point mutation (43)
tivities of hybrids examined 2, 5, and 15 weeks aRer cloning, respec- accumulate in mtDNA during aging. In fact, mammalian
tiveiy. Modal chromosome numbers of pO-HeLa cells, TIG102, mtDNA evolved 10 times faster thansingle-copy nuclear DNA
Hybrids-1, -2, and -3 were 50,42,89, SO, and 77, respectively.
(44).On the other hand, many studies have failed to demon-
strate a~umulationof somatic mutations in mtDNA popula-
activity in thehybrids was paralleled by gradual chromosome tions of human individuals. For example, the mtDNA m u ~ t i o n
loss (Fig. 6). Therefore, these observations suggest that nuclear levels within single individuals have been shown to be ex-
genes of HeLa cells responsible for the restoration of COX ac- tremely limited 145,461, and thusabout 10l6mtDNA molecules
tivity are located separately on several chromosomes. within an individual are considered to be almost identical to
one another (47). Even in thecase of the heteroplasmic mtDNA
DISCUSSION molecules in a patient with mitochondrial myopathy, encepha-
In this work, we found an in vivo age-related decrease of lopathy, lactic acidosis, and stroke-like episodes, the mutation
mitochondrial energy production in cultured human skin fibro- was not somatic, and no other mutations were observedamong
blasts obtained from 16 normal donors of various ages (0-97 them (48). Bodenteich et al. (49) detected no age-related accu-
years) and showed that this phenotype was due to accumula- mulation of somatic mutations in mtDNA of human retina,
tion of nuclear-recessive somatic mutations, and not due to a even though this tissueis postmitotic and is constantly exposed
decrease in the copy number of mtDNA moleculesor the accu- to UVlight in life. Moreover, Monnat and Realy (50)observed
mulation of various kinds of somatic mutations in mtDNA in no accumulation of somatic mutations in the mtDNA of differ-
fibroblasts of aged persons. ent tissues within one individual. Furthermore, no somatic
A similar decrease of mitochondrial respiratory activity re- mutations were induced in the mtDNA of HeLa cells by treat-
lated to donor age has been observed in human muscle (4-6) ment with chemical carcinogens(51). However, approaches us-
and liver (7). The amount of the common large scale deletion ing mtDNA sequence analysis could not provide direct evidence
mutant AmtDNA4977,which was first found to accumulate pre- of whether accumulation of somatic mutations in mtDNA plays
dominantly in skeletal muscles of patients of mitochondrial a causal role in aging andlor carcinogenesis.
myopathy (29,30), was shown to increase with age in human Cytoplasmic mtDNAtransfer techniques should resolve this
skeletal muscle, liver,and brain(8-11). However, its content in issue by testing whether mtDNAgenotypes and the phenotypes
old subjects was very small (0.005~.1%) compared with that in related to carcinogenesisor aging can be co-transferred to other
patients with m i t o c h o n ~ a myopathy
l (20-90%).Since reduc- cells. "hese techniques have been used successfully for demon-
tion of COX activity was heterogeneous in muscle fibers of aged strating thataccumulation of mtDNA mutations isresponsible
o nAmtDNA4977was also het- for mitochondrial dysfunction observed in mitochondrial dis-
subjects (4, 5) and a c c ~ ~ a t i of
erogeneous in brain tissues (31, 321, focal accumulation of a eases (20, 52-54). These techniques have also shown that
small amount of the mutant mtDNA possiblyinduced age-re- mtDNA mutations are not involved in the expression of tumori-
lated mitochondrial dysfunction in limited regions of these or- genicity in HeLa (27, 55) or rat glioma (56) cells and that the
gans. It is also possible that, in addition to this common dele- phenotype of carcinogen-induced tumorigenicity expressed in
tion, the accumulation of various other unidentified somatic mouse skin fibroblasts is transmitted through the nuclear ge-
mutations in the mtDNA population causes the age-related nome only, not through the mitochondrial genome (57). In this
mitochondrial dysfunction (33, 34). work, intercellular transfer of the mitochondrial genome from
On in vivo aging of normal human skinfibroblasts, however, fibroblasts of aged subjects to pO-HeLa cells provided convinc-
donor age-related reduction of COX activity was not heteroge- ing evidence that the in vivo age-related mitochondrial dys-
neous in individual fibroblasts from the same donors, and function found in human skin fibroblasts is not controlled by
transfer of their mtDNA to po-HeLa cells showed that the fi- the mitochondrial genome.
broblast mtDNA in aged donors was functionally intact. More- In contrast, intercellular transfer of HeLa nuclear genome to
over, AmtDNA4977was not detected in any cybrid clones or in fibroblasts from an aged donorclearly showed that thereduced
mtDNA donor fibroblasts by PCR, which can detect 0.002%of COX activity in the aged fibroblasts can be restored by intro-
AmtDNA4977.Large scale deletion mutant mtDNA molecules duction of HeLa nuclear genome, suggesting that human age-
 Functional Integrity of Human
mtDNA
from
related mitochondrial dysfunction was inherited in a nuclear-
recessive way. Moreover,these cells subsequently began to lose
the restored COX activity during cultivation in association with
gradual chromosome loss. Accordingly,nuclear genes of HeLa
cells, which were responsible for the restoration of COX activ-
ity, appeared to be localized separately on several chromo-
somes. These observations support the idea that accumulation
of nuclear-recessive somatic mutations ingenes located on vari-
ous chromosomes is responsible for the age-related mitochon-
drial dysfunction observed in human skin fibroblasts kf. Fig.
1). This age-related phenotype appears to be quite different
from the age-related mortality phenotype observed in human
diploid fibroblasts, where mortality is inherited in a nuclear-
dominant way, because hybrids obtained from fusion of normal
human fibroblasts with various transformed human cell lines
exhibited limited potential for cell division (17, 58).
To extend our conclusion to ~ s t m i ~ t ihighly
c, oxidative or-
gans such as the brain and heart, we are now investigating
whether these organs also show the age-related mitochondrial
dysfunction and whether mtDNA and the phenotype in these
organs can be co-transferred to pO-HeLa cells.
REFERENCES
1. Linnane, A. W., Marzuki, S., Ozawa, T. and Tanaka, M. (1989) Lancet 3,
612-645
2. Miquel, J. (1991)Arch. Gerontol. Geriatr. 12, 99-117
3. Wallace, D. C. (1992) Science 266,628-632
4. Muller-Hiicker, J. (1989) Am. J. Pathol. 134, 1167-1173
5. Muller-Hacker, J. (1990) J . Neurol. Sci. 100, 14-21
6. Trounce, I., Byme, E., and Manuki, S . (1989) Lancet 1,637-639
7. Yen, T.-C., Chen, Y.-S., King, K.-L., Yeh, S.-H., and Wei, Y.-H. (1989)Biochern.
Biophys. Res. Cornmun. 165,994-1003
8. Ikebe, SA., b ak a ,M., Ohno, K , Sato, W., Hattori, K., Kondo, T., Mizuno, Y.,
and Ozawa, T. (1990) Biochem. Biophys. Res. Comrnun. 170,1044-1048
9. CortOpassi, G.A,, and Amheim, N. (1990) Nucleic Acids Res. 18, 6927-6933
10. Corral-Debrinski, M.,Stepien, G., Shoffner,M. M., Lott, M. T., Kanter, H., and
Wallace, D. C. (19911 J. A m Med. Assoc. 266, 1812-1816
11. Yen,T.-C., Su.,J.-H., King, K.-L., and Wei.Y.-H. (1991) Biochern. Biophys. Res.
Cornmun. 178,124-131
12. Gadaleta, M.N., Petmzella, V., Renis, M., Fracasso, F., and Cantatore, P.
(19901 Eur. J. Biochem. 187,501-506
13. Attardi, G., King, P., Chomyn, A. and Polosa, P. L. (1991) Progc Neuropathol.
7,7692
14. Clayton, D. A. (19841Annu. Rev. Biochem 63,573494
15. Attardi, G.,and &hats, G.(1988) Annu. Rev. Cell B i d 4 , 2 8 9 4 3 3
16. Hayfiick, L. (1965) Exp. Cell Res. 37,614436
17. Goldstein, S . (1990) Science 248,1129-1133
18. Coldstein, s., Ballantyne, S . R., Robson, A. L., and Moerman, E.J. (1982) J.
Cell. Physid. 112,419-424
19. Hayashi, J.-I., Yonekawa, H., Watanabe, S., Nonaka, I., Yoshida Momoi, M.,
Kagawa, Y., and Ohta, S . (1991)Progr. Neuropathoi. 7,93-102
20. Hayashi, J:I., Ohta, S . , Kikuchi, A,, Takemitsu, M., Goto, Y.-i., and Nonaka, I.
(1991) Proc. Nati. Acad. Sci. U. S.A 88, 10614-10618
21. Hayashi, J.-I., Yonekawa, H., Murakami, J., Tagashira, Y., Pereira-Smith, 0.
M., and Shay, J. W. (1987) Exp. Cell Res. 172, 218-227
22. Hayashi, J.-I., Werbin, H., and Shay, J. W. (1986) Cancer Res. 46, 4001-4006
Aged
Subjects 6883
23. Mariottini, P., Chomyn, A,, Riley, M., Cottrel, B., Doolittle, R. F., and Attardi,
G. (1986) Proc. Natl. A d . Sci. U. S.A 83,1563-1567
24. Miyahayashi, S . , Narisawa, K., Iinuma, K., Tada, K., Sakai, K, Kobayashi, K,
Kobayashi, Y., and Morinaga, S . (1984) Brain Deu. 6 , 3 6 2 3 7 2
25. Seligman, A. M., Karnovsky, M. J.,Wasserkrug, H. L., and Hanker, J. S.(1968)
J. Cell. Eiol. 38, 1-14
26. Sun,A. S.,Aggarwal, B. B., and Packer, L.(1975)Arch.B k h e m . Biophys. 170,
1-11
27. Shmookler Reis, R. J., and Goldstein, S . (1983) J. Biol. Chern. 268.9078-9085
28. Gadaleta, M. N., Rainaldi, G., Lezza, A. M. S., Milella, F., Fracasso, F., and
Cantatore, P. (1992) Mutat. Res. 276,181-193
29. Holt, I. J., Harding, A. E., and Morgan-Hughes, J. A. (1988) Nature 381,
717-719
30. Schon, E.A,, Rizzuto, R., Moraes, C. T., Nakase, H., Zeviani, M., and DiMauro,
s. (1989)science 244,346-349
31. h n g , N. W., Hinton, D. R., Cortopassi, G., and Amheim, N. (1992) Nature
Genet. 2,318-323
32. Corral-Debrinski, M., Horton, T., Lott, M. T., Shoffner, J. M., Flint Beat, M.,
and Wallace, D. C. (1992)Nature Genet. 2,324-329
33. Cortopassi. G. A., Shibata, D., Soong, N.-W., and Amheim, N. (1992) Pmc.
Natl. Acad. Sei. U. S. A. S S, 7370-7374
34. Linnane, A. W., Zhang, C., Baumer, A., and Nagley, P. ( 1992) Mutat. Res. 275,
195-208
35. Rirtig, A,, Cormier, V., Blanche, S., Bonnefont, J.-I?, Ledeist, F., Romero, N.,
Schmitz, J., Rustin, P., Fischer, A., Saudubray, J.-M., and Munnich, A.
(1990)J. CZin. Invest. 86,1601-1608
36. M e n , J. A., and Coombs, M. M. (1980) Nature 287,244-245
37. Backer, J. M.,and Weinstein, I. B. (1980) Science 2oS, 297-299
38. Niranjan, B. G.,Bhat, N. K., and Avadhani, N. C . (1982) Science 215,73-75
39. Shay, J. W. and Werbin, H. (1987) Mutat. Res. 186,149-160
40. Hayakawa, M., Toni, K , Sugiyama, S., Tanaka, M., and Ozawa, T. (1991)
Biochem. Biophys. Res. Cornmun. 179, 1023-1039
41. Sugiyama, S., Hattori, K., Hayakawa, M., and Ozawa, T. (1991) Biochem.
Bwphys. Res. Cornmun. 180,894-899
42. Zhana, C., Baumer, A,, Maxwell, R. J., Linnane, A. W., and Napley, P. (1992)
FEBS Lett. 297,34-38
43. Mimscher, C., Rieger, J., Muller-Hocker, J., and Kadenbach, B. (1993) FEBS
Lett. 317, 27-30
44. Brown, W. M., George, M., Jr., and Wilson, A. C. (1979) Pmc. Natl. Acad. Sci.
U. S. A. 76. 1967-1971
45. Brown, W. M.'(1980)-&x. Natl. Acad. Sei. U. S. A. 77,3605-3609
46. Monnat, R. J., Jr., and Loeb, L.A. (1985) Proc. Natl. Acad. Sci. U. S. A. 88,
2895-2899
47. Cann, R. L., Stoneking, M., and Wilson, A. C. (1987) Nature 3 2 6 , 3 1 4 6
48. Kobayashi, Y., Momoi, M. Y., Tominaga, K, Shimoizumi, H., Nihei, K, Yanag
isawa, M., Kagawa,Y., and Ohta,S. (1991)Arn.J. Hum. Genet. 43,590-599
49. Bodenteich, A,, Mitchell, L. G., and Menil, C. R. (1991) Gene 108,305-310
50. Monnat, R. J., Jr., and Realy, D. T. (1986) Gene 43,205-211
51. Mita, S., Monnat, R. J., Jr., and Loeb, L. A. (1988) Cancer Res. 48,45784583
52. Chomyn, A,, Meola, G., Bresolin, N., Lai, S . T., Searlato, G., and Attardi, G.
(1991) Mol. Cell. Biol. 11, 223E-2244
53. King, M. P., Koga,Y., Davidson, M., and Schon, E. A. (1992)M d . Cell. Biol. 12,
480-490
54. Chomyn, A., Martinuzzi, A. Yoneda, M.,Daga, A., Hurko, 0.. Johns, D., h i , S.
T., Nonaka, I., Angelini, C., and Attardi, G. (1992) Proe. Natl. Acad. Sei.
U. S. A. gS, 4221-4225
55. Hayashi, J.-I., Takemitsu, M., and Nonaka, I. (1992) Somatic Cell Mol. Genet.
18,123-129
56. Hayashi, J.-I., Tagashira, Y., Watanabe, T., and Yoshida, M. C. (1984) Cancer
Res. 44,3957-3960
57. Hayashi, J.-I., Yonekawa, H., and Tagashira, Y.(1989) Cancer Res. 49, 4715-
4720
58. Pereira-Smith,0.M., and Smith, J. R. (1988)Proc. Natl. Acad. Sci. U. S. A 86,
6042-6046