Genotypic and phenotypic changes in exhaustively grown cell lines from mitochondrial cytopathy...

ABSTRACT: Understanding the pathobiology of mitochondrial (mt) DNAdiseases involves both characterization of the effects of individual mutationson respiratory function and elucidation of the changes in mutation load anddistribution (energy mosaicism) over serial cell generations. Whether a givenmutation is stably maintained, or increases or decreases with cell growth, isone of the determinants as to whether a particular tissue will be affected byoxidative phosphorylation failure. In this study, we correlated mt genotypewith biochemical phenotype in myoblasts from patients with pathogenicmtDNA mutations. The dominant process detected was a progressive elimi-nation of mutant mtDNA genomes concomitant with an improvement in re-spiratory chain activity, suggesting that energetically normal cells have agrowth advantage over those with a high mutation load. We propose that thiselimination is by biased distribution of wild-type mtDNA to daughter cells,and that a similar mechanism could operate in vivo and contribute to boththe clinical expression of mt disease and the maintenance of a predomi-nantly wild-type mt genome pool across generations. © 1998 John Wiley &Sons, Inc. Muscle Nerve 21: 599–609, 1998Key words: mitochondrial cytopathies; mitochondrial DNA mutations; myo-blasts; in vitro proliferation; disease expression

GENOTYPIC AND PHENOTYPICCHANGES IN EXHAUSTIVELY GROWNCELL LINES FROM MITOCHONDRIALCYTOPATHY PATIENTS

NURJATI C. SIREGAR, MD, 1 M.J. BERNADETTE JEAN-FRANÇOIS, PhD, 2*

ROZANNE B. BLOK, PhD, 2 and EDWARD BYRNE, DSc, MD, FRACP 2

1 Department of Medicine, St. Vincent’s Hospital, Fitzroy, Victoria, 3065, Australia2 Melbourne Neuromuscular Research Centre, St. Vincent’s Hospital, Fitzroy,Victoria, 3065, Australia

Received 19 June 1997; accepted 17 November 1997

In mitochondrial (mt)DNA disease, the develop-ment of respiratory failure in a given cell populationdepends on the pathogenic potential of the indi-vidual mtDNA mutations and on the changes in mu-tation load with serial mitochondrial and cellulargenerations. Fixed postmitotic cell populations (i.e.,central nervous system, are primarily involved in thedisease process, but tissues which have a regenerativecapacity (i.e., skeletal muscle and liver) are also in-volved in many cases. In this study, we selected ahuman myoblast model to study mtDNA changesand phenotypic consequences over a number of celldivisions. This model allows us to correlate genotypewith phenotype in serial generations, to explore the

role of mt failure in cells grown to exhaustion (Hay-flick phenomenon),20 and to determine the popula-tion kinetics of mutant and wild-type mtDNA in di-viding cells.

mtDNA diseases typically exhibit heteroplasmy,whereby wild-type and mutant DNA coexist in vary-ing proportions, and energy mosaicism, whereby bio-energetically, cells with predominantly wild-type ge-nomes and cells with oxidative phosphorylation(OXPHOS) deficiency manifest by cytochrome c oxi-dase (COX) deficiency and accumulation of mutantgenomes in the same tissue. In both chronic progres-sive external ophthalmoplegia (CPEO), where 40–50% of patients have a large multigene deletion,22,31

and mitochondrial encephalomyopathy, lactic acido-sis, and strokelike (MELAS) syndrome, where 80%have a point mutation at nucleotide 3243 (A to G) intransfer (t)RNALeu(UUR) gene,17 these phenomenaof heteroplasmy and energy mosaicism are seen.10,32

In vitro studies into the expression of mt cytopa-

*Correspondence to: Dr. M.J. Bernadette Jean-FrancoisContract grant sponsors: National Health and Medical Research Councilof Australia; AusAID

CCC 0148-639X/98/050599-11© 1998 John Wiley & Sons, Inc.

Mitochondrial Cytopathy Myoblasts MUSCLE & NERVE May 1998 599

thies have mainly focused on the fusion of enucle-ated patient fibroblasts to immortalized cell linesrendered mtDNA-less after prolonged treatmentwith ethidium bromide.25 These cybrids have shownthat in MERRF (myoclonic epilepsy and ragged-redfibers) and MELAS derived transformants with >95%mutant mtDNA, the associated defects in respirationand protein synthesis were transferred to the cy-brids.8,26 Importantly, functional complementationwas achieved provided the cell line consisted of atleast 5% wild-type mtDNA.8,9 A shift from the wild-type to pure mutant genotype in heteroplasmic cy-brids has also been observed, indicating an intracel-lular replicative advantage by the mutant mtDNA.2,41

While results obtained from cybrid studies areuseful in deriving the phenotypic effects of indi-vidual mtDNA mutations on respiration, several as-pects in the understanding of mt cytopathies are un-resolved. It is known that energy mosaicism in vivo,reflected by uneven distribution of mutant and wild-type mt genomes in cells, leads to a fairly broadrange of bioenergetic outcomes. However, little isknown about its behavior (expression and distribu-tion of mtDNA mutations), in particular, how itchanges with increasing mt and cell generations andthe resultant effect on overall organ function.

Earlier studies in fibroblast cultures have shownthat deletions were eliminated after several pas-sages6; more recently, it was shown that a novel mu-tation in the tRNALeu(CUN), present in high propor-tions in two separate skeletal muscle biopsies from apatient, was not present in the satellite cells.11,14

However, the mutation was also absent from blood,suggesting a new germline mutation.

In this study, the key aim was to carry out a de-tailed analysis of the behavior of two major patho-genic mt mutations in myoblast cultures by correlat-ing mutation load with respiratory chain activity atdifferent points in the cell growth cycle. The muta-tions studied were the MELAS nt3243 A to G muta-tion and the CPEO 4.9-kb common deletion. Newfindings to emerge from this work include: (1) elimi-nation of mitochondrial mutation from cells divid-ing in culture; (2) delineation of the time kineticsfor this process, indicating a more rapid rate ofelimination for the MELAS point mutation than thecommon deletion; (3) demonstration of an improve-ment in respiratory chain activity with mutationelimination; and (4) demonstration that the Hay-flick phenomenon applies to myoblasts, and that it isnot influenced by respiratory chain phenotype.

The elimination of mtDNA mutations in dividingcells is of special interest, as it has implications re-garding in vivo pathogenicity: the growth character-

istics of an individual tissue may be a determinant inthe development of OXPHOS failure; in addition,the capacity of dividing cells to readily eliminate mu-tant cells will influence the molecular genetics ofmtDNA mutations over time within a given subjectand across generations.

MATERIALS AND METHODS

Clinical History of Patients. Mitochondrial CytopathyPatients. MELAS1 is a 48-year-old female with epi-lepsy, dementia, profound deafness, focal neurologi-cal deficits, cognitive impairment, and diabetes. Acerebral computed tomography (CT) scan showeddense basal ganglial calcifications, cerebral and cer-ebellar atrophy, focal atrophy of the left posteriorregion, and a small hypodensity in the left parietallobe. She is a member of a family with a documentedhistory of MELAS.28 Histologically, a muscle biopsyshowed ragged-red fibers (RRF), while mtDNAanalysis revealed the presence of the MELAS nt3243mutation.

MELAS2 is a 15-year-old female with sensorineu-ral hearing deficit since the age of 4 years, growthdeficit, and stroke. She had persistent headaches,nausea, vomiting, blurred vision with left hemiano-pia, and transient right hemiparesis. A magneticresonance imaging (MRI) scan revealed a sizeablearea of edema in the right occipital region which wasnot in a vascular territory. She was diagnosed withMELAS and treated with CoQ10 and prednisolonefor a few months. A second MRI scan showed com-plete resolution of the original lesion. Histologically,a muscle biopsy confirmed a moderate number ofRRF and COX-negative fibers, while mtDNA analysisrevealed heteroplasmic expression of the MELASnt3243 mutation.

MELAS3 is a 19-year-old male with a history ofgeneralized seizures, myoclonus, and frequent at-tacks of photopsia spreading across the right fieldwith partial loss of vision lasting for several minutes.With time, the attacks became more frequent andwere precipitated by exertion. An MRI of the brain,an electroencephalogram (EEG), and an echocar-diogram were normal. A muscle biopsy revealed nu-merous RRF but no COX-negative fibers. Serum lac-tate was elevated (3.7 mmol/L; normal: 0.4–3.2mmol/L). A diagnosis of MELAS was made, and hewas given treatment for epilepsy (sodium valproate,vigabatrin, lamotrigine, and carbamazepine) as wellas CoQ10 and prednisolone. mtDNA analysis of themuscle biopsy revealed the presence of the MELASnt3243 mutation.

CPEO1 is a 16-year-old female with marked ex-ternal ophthalmoplegia, moderate bilateral ptosis,

600 Mitochondrial Cytopathy Myoblasts MUSCLE & NERVE May 1998

and fatigue. Histologically, a muscle biopsy revealedRRF and COX-negative fibers. She was diagnosed asCPEO and given CoQ10 treatment. DNA studies(Southern blot) identified the heteroplasmic expres-sion of the 4.9-kb mtDNA common deletion.23

CPEO2 is a 57-year-old female with a 25-year his-tory of fatigue and a 10-year history of progressiveptosis and external ophthalmoplegia. She developedaspiration before presentation. A muscle biopsy re-vealed RRF and COX-negative fibers, indicating mi-tochondrial cytopathy. DNA studies (Southern blot)revealed the presence of the mtDNA common dele-tion in a heteroplasmic manner.23

Disease–Control Patients. Skeletal muscle biopsies(m. quadriceps femoris or m. vastus lateralis) wereobtained from 9 control patients investigated formuscle pain, weakness, or fatigue, where muscle bi-opsy histology revealed either no abnormality ornonspecific type IIb fiber atrophy. Four patients suf-fered from fatigue or myalgia, 2 had a single episodeof unexplained rhabdomyolysis, 2 had mild proxi-mal weakness with no myopathic features on biopsy,and 1 had epilepsy. Myoblast cell lines were derivedfrom these biopsies and treated under similar con-ditions as for the mt cytopathy patient-derived celllines. Disease–control patients will be referred to ascontrols in this study.

Myoblast Cell Lines. Skeletal muscle biopsies wereobtained from mt cytopathy patients and controlsubjects with approved informed consent and in ac-cordance with the guidelines set by the HREC (Hu-man Research Ethics Committee) of St. Vincent’sHospital. Myoblast cultures were established fromsatellite cells essentially following the method of Bis-choff,4 with preplating for 30 min to minimize con-tamination by fibroblasts. They were grown in Ham’sF-12 medium (GIBCO) containing 20% fetal calf se-rum (TRACE),5 antibiotics, and 2.5 ng/mL of basicfibroblast growth factor (bFGF) (Promega),37 pas-saged at 70% confluence following treatment with0.125% wt/vol trypsin (CSL), and replated at a den-sity of 1 × 104 per cm.2 Cultures were passaged up to12–15 times to growth exhaustion. Cell populationdoubling level was calculated for each cell line atevery passage number from the formula: NH/NI = 2X,where NH is the number of cells harvested and NI isthe number of cells subcultivated.12 Data were statis-tically analyzed by the Pearson correlation method(Statistica 5.0, Statsoft, Inc.). Samples were taken atearly (P5 or P6), mid (P10 or P11), and late passage(P15 or P16) numbers for genetic and biochemicalstudies. Selected cultures were stained with Desmin(DAKO) and counterstained with Harris hematoxy-

lin to estimate the degree of contamination by fibro-blasts.

Enzyme Complex Assays. These were carried outon mitochondria isolated from myoblasts (usually10–18 × 106 cells) after sonication in isolation me-dium.34 Complex I (NADH–ubiquinone oxidore-ductase, EC 1.6.5.3),18 complexes II–III (succinate–cytochrome c oxidoreductase, EC 1.3.5.1 and EC1.10.2.2), and complex IV (cytochrome c oxidase,EC 1.9.3.1)36 were measured spectrophotometricallyin a GBC spectrophotometer. Assays were performedat least in duplicate and where possible (in 9 of 16cell lines), from two independent mitochondrial iso-lations at the early and late stages of growth. Forevery experiment, mitochondria from at least onecontrol cell line were also isolated and included inthe enzyme assays. Skeletal muscle (vastus) mito-chondria, stored in aliquots at −80°C and originallyobtained from a patient undergoing orthopedic sur-gery and with no known history of mitochondrialdisease, were also included to check assay reproduc-ibility and spectrophotometer calibration (data notshown). The activity of citrate synthase, a matrix en-zyme, was also determined in all experiments as aninternal standard (data not shown). The activity ofcomplexes II–III and IV were determined on freshlyisolated mitochondria; complex I activity was per-formed on frozen and thawed (×1) mitochondria.

DNA Studies. Detection and Quantification of theMELAS nt A to G 3243 Point Mutation by polymerasechain reaction–restriction fragment length polymorphism(PCR-RFLP). Total DNA extracted from the musclebiopsy (10–20 mg) or from myoblasts at various pas-sage numbers (5 × 106 cells) was amplified by PCR ina 50-µL reaction volume containing 20 pmol each ofprimers L316 (nt3160–nt3179) and H330 (nt3306–nt3272) and 1.25 U Taq polymerase; initial denatur-ation was set at 95°C for 2 min and was followed by24 cycles [95°C for 30 s and 65°C for 2 min (ITC 100;Bartelt Instruments)]. To assess the extent of restric-tion digestion by HaeIII in subsequent procedures, 8pmol each of primers L498 (nt4981–nt5003) andND2H (nt5482–nt5459) were also included to gen-erate a PCR product with a single HaeIII site (nt5261;data not shown). Ten microcuries of a32P-deoxycytidine triphosphate (dCTP) was added dur-ing the denaturation step of the final PCR cycle(95°C, 5 min; 65°C, 2 min). The PCR-amplified frag-ment (10 of 50 µL) was then digested with HaeIII (20U, Promega) in a 20-µL reaction at 37°C for 2 h. The


digestion products were electrophoresed (8% non-denaturing polyacrylamide gel) to reveal a 140-bp-long wild-type band and/or mutant bands 83 bp and57 bp long, respectively; the ratio of wild-type to mu-tant bands was estimated by laser densitometry (Per-sonal Densitometer SI; Molecular Dynamics) follow-ing autoradiography at room temperature (RT). Apositive control clone harboring the nt3243 muta-tion (kindly prepared by Miss S. Katsabanis) and anegative control were also included in each experi-ment.

Detection and Quantification of the CPEO CommonDeletion by Southern Blot. Total DNA was extracted asabove from the muscle biopsy or myoblasts at variouspassage numbers, and 1–2 µg was digested with 10Uof BamHI, electrophoresed on a 0.6% wt/vol agarosegel, transferred onto a nylon membrane (HybondN+, Amersham), and hybridized as previously de-scribed.23 The mtDNA probe was labeled with Di-goxigenin (whole mt genome, DIG; BoehringerMannheim),23 or a32P-dCTP (nt1581-nt5482; Amer-sham). The DIG-labeled bands were colorimetricallydetected with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indoyl phosphate (X-phosphate)for 1–3 h at RT. Autoradiography was at RT, andmtDNA digestion products were semiquantitativelyestimated as above.

Detection of the CPEO Common Deletion by PCR. To-tal DNA from muscle or myoblasts was amplified bya semicompetitive PCR method in a 50-µL reactionvolume with the primers L7901 [(nt7901–nt7920),40 pmol] and H1365 and C2R1 [(nt13631–nt13650and nt8521–nt8540), 20 pmol each]; initial denatur-ation was at 95°C for 2 min followed by 30 cycles withdenaturation at 95°C for 30 s, annealing at 55°C for1 min, and extension at 72°C for 1 min.

DNA Sequencing of PCR Product Harboring MELASnt3243 Mutation. Total DNA extracted from myo-blasts at various passage numbers was amplified byPCR in 100-µL reactions containing 40 ng of tem-plate and the primers B1F1 (nt2826–nt2849) andH388SQ (nt3388–nt3363). After purification of thePCR products (Wizard PCR preps DNA purificationsystem, Promega), DNA sequencing was performedby the dideoxy termination method (fmol kit, Pro-mega) using 50 ng of template DNA, 1.5 pmol ofL316 primer (see above) labeled at the 58 end withg32P-deoxyadenosine triphosphate (dATP) (10mCi/3000 mmol), and 5 U of Sequencing grade TaqDNA polymerase (Promega). Conditions for thePCR were: initial denaturation at 95°C for 2 minfollowed by 30 cycles of denaturation at 95°C for 30s, annealing at 55°C for 30 s, and extension at 72°Cfor 30 s. Aliquots (2–3 µL) were electrophoresed on

6% acrylamide, 7 mol/L urea gels, and the vacuum-dried gels were exposed to X-ray film (BioMax, KO-DAK) for 2–4 h at RT.

RESULTS

In Vitro Growth Characteristics. Myoblast cell linescould be established from skeletal muscle biopsiesirrespective of the age of donor and passaged up tobetween 15 and 17 times (control group); supple-mentation of the Ham’s F12 medium with bFGF at aconcentration of 2.5 ng/mL was found to signifi-cantly enhance the rate of growth in culture. In allcultures stained with desmin and counterstainedwith hematoxylin, myoblasts made up 95% or moreof the cell population, suggesting that under theconditions used, fibroblast growth does not overtakemyoblast growth with cell division (data not shown).

Cell population doubling level (CPDL) was de-termined at every passage number after trypsin treat-ment of the cells. Cultures were initially inoculatedat a density of 1 × 104 per cm.2 After establishment ofthe cell lines at about passage number 3, an inversecorrelation was observed between CPDL and passagenumber for both the control group and the mito-chondrial cytopathy groups (Fig. 1a). This decreasewas statistically significant for the control andMELAS groups [P = 0.04 (control), P = 0.006(MELAS), and P = 0.14 (CPEO)] and appeared mostpronounced in MELAS patients, where an averagedecline/day of 0.032 was observed, followed by theCPEO group (0.026) and the control group (0.01).

When CPDL was plotted against donor age attime of muscle biopsy, an inverse direct correlationwas obtained for the control group, implying thatthe older the donor, the greater the apparent gen-eration time of the myoblasts in culture (Fig. 1b).MELAS2 and CPEO1, both aged 16 and severely af-fected clinically, were found to have a low CPDL of0.14 and 0.15, respectively, suggesting that the na-ture and clinical severity of the syndrome are alsosignificant in determining myoblast generation time.

Activity of the Respiratory Chain (RC). In general,the changes observed in RC activity with in vitro cellproliferation followed a trend in the same directionsuch that an increase in activity of complexes I andIV was seen in the MELAS and CPEO derived myo-blasts; the biggest increase was in complex I inCPEO1, where a 625% increase was recorded (Fig.2a). MELAS1 displayed the largest increase in com-plex IV activity (395%, Fig. 2b). However, CPEO2displayed an increase in complex I and a decrease incomplex IV activity; this patient was older than otherpatients at time of muscle biopsy and had a lower


starting level of complex IV activity (25%). She wasthe only cytopathy patient where complex I and IVactivities showed discordant findings in late cultures,and the significance of this observation is not clear;it may be that in a small subset of patients, complexactivities in late culture may show divergent changes,but it is clear from the data here that in general, thealterations in complex I and IV activity are similar. Inthe control group, changes in activity were much lessmarked, with a small improvement in activity insome cases and a decline in others (Fig. 2a, b); over-all, an average decline of 10% in complex I activityand 29% in complex IV activity was observed. Mea-surement of the activity of complexes II–III revealedthat there were no significant variations in any of thegroups with cell proliferation (data not shown).

Irrespective of the enzyme complex assayed, awide range in initial activity was obtained for thecontrol group and the mitochondrial cytopathygroup; for instance, complex I activity in the controlgroup ranged from 23.6 to 60.4 nmol/min/mg and

in the cytopathy group from 12.6 to 56 nmol/min/mg. Such variations could reflect age and/or physi-ological differences. To circumvent this, enzyme ac-tivities at the mid and late stages of growth areexpressed as a percentage of the initial activity atearly passage.

In parallel with an increase in enzyme complexactivity, there appeared to be a decrease in mito-chondrial protein synthesis with serial passaging(data not shown). When equal amounts of proteinwere loaded on a gel, changes in the sodium dodecylsulfate/polyacrylamide gel electrophoresis (SDS-PAGE) pattern of [35S] methionine-labeled mito-chondrial translation products were noted such thatan overall decrease in the intensity of bands was ob-served, particularly with the larger molecular weightsubunits for both the control and cytopathy groups.However, noted variations in the pattern for indi-vidual subjects within a group together with the tech-nical difficulties associated with labeling small cellnumbers confirm the need for further studies.

FIGURE 1. Relationship between cell population doubling level and in vitro cell proliferation (a) and donor age at time of skeletal musclebiopsy (b). (a) CPDL/day vs. passage number in mitochondrial cytopathy patients [MELAS (j) and CPEO (m)] and in controls (d). TheMELAS group consisted of 3 patients, the CPEO group consisted of 2, while the control group had 9 patients. For each patient group,the average CPDL value together with the standard error of the mean at every second passage number was calculated, but is illustratedfor the control group only for ease of visualization. For every patient, two determinations were carried out and the average calculated. Datawere statistically analyzed by the Pearson correlation method. A computer-generated regression curve of best fit is included for eachgroup showing an inverse correlation between CPDL/day and passage number; the decrease was statistically significant for the controland MELAS groups only (P = 0.04* for control; P = 0.006* for MELAS; and P = 0.14 for CPEO). The solid line represents the control group,the interrupted line the CPEO, and the dotted line the MELAS patients. *Statistically significant. (b) CPDL/day vs. donor age at time ofmuscle biopsy for mt cytopathy patients (m, j) and control patients (d). For each individual, the average of two measurements is shown.A computer-generated regression line of best fit for the control group is shown (P = 0.69).


DNA Studies. Analysis of the mitochondrial geno-type for the CPEO group was carried out at three ormore different passage numbers by Southern blot-ting with a mt probe encompassing the entire ge-nome and by a semicompetitive, nonquantitativePCR for the wild-type and mutant fragments. TheMELAS group was analyzed by PCR-RFLP and bydirect sequencing of PCR products (Fig. 3).

CPEO Patient Group. Figure 3a illustrates the re-sults of a Southern blot analysis of a muscle biopsyand myoblasts at various passage numbers for

CPEO1, CPEO2, and a control patient; Figure 3bshows PCR results for CPEO1 at passage numbers 9,10, 11, and 14. Estimates of the percentage of wild-type to deleted mtDNA strains obtained by densito-metric scan of the Southern blot are depicted inFigure 4. A gradual elimination of the mutant spe-cies was observed with cell division: in CPEO1, thepercentage of wild-type mtDNA (16.5-kb band) pre-sent in the muscle biopsy was 20%; at P10 it was 70%,and by P14, the common deletion (11.5-kb band)was no longer detectable by Southern blotting or

FIGURE 2. Complex I (NADH ubiquinone oxidoreductase) and complex IV (cytochrome c oxidase) activities in mt cytopathy and controlpatients. Complexes I and IV activities were determined spectrophotometrically, and are expressed for each patient as a percentage ofthe initial activity at early passage to allow for the wide range in initial activity (as explained in the Results). Duplicate measurements weremade for each patient, and the mean and standard error of the mean are shown except for the controls. Complex IV activity for 7 controlpatients is depicted, whereas complex I activity for only 5 control patients is shown as a result of an inadequately low number of cells beingharvested at the later stage of growth. The same applies to patient CPEO2, where by P12, the cells were nearly grown to exhaustion.(a) Complex I activity. (b) Complex IV activity.


PCR and had therefore been eliminated. The PCRdata (Fig. 3b) indicate that the deletion is effectivelyeliminated after passage 11 and before passage 14.In CPEO2, the percentage of mutant mtDNA in themuscle biopsy was 29%, by P5 it was 22%, and by P9it was no longer detectable by Southern blotting orPCR (data not shown). It is noteworthy that for the

CPEO patients studied here, the ratio of wild-type tomutant mtDNA, in the muscle biopsy and at P0 andP1, did not alter by as much as has been previouslyreported for myoblasts and fibroblasts with the com-mon deletion.6,39

MELAS Patient Group. The results of the PCR-RFLP and PCR-DNA sequencing for the fragment

FIGURE 3. Expression of wild-type and mutant mtDNA species in CPEO (a,b) and MELAS (c,d) patients depicting mutation eliminationwith cell division. (a) Southern blot analysis of common deletion in CPEO patients. Total cell DNA from muscle or myoblasts [at musclebiopsy (M) and indicated passage numbers] was digested with BamHI and following transfer, hybridized with a a32P-dCTP-labeledmtDNA probe (myoblast-derived DNA samples) or a DIG-labeled mtDNA probe (muscle-derived DNA samples). The 16.5-kb band refersto the wild-type species, the 11.5-kb to the deleted species. Molecular weight markers were DIG-labeled; CTL refers to control. (b) PCR(semicompetitive, nonquantitative) analysis of common deletion in CPEO1 showing the elimination of deleted species between P11 andP14. Total cell DNA from myoblasts (P9–P14 for CPEO1 and P0 for control) was amplified by PCR with the three primers L7901 (40 pmol),and H1365 and C2R1 (20 pmol each). Amplified product from wild-type and deleted species is 636 and 772 bp long. (c) PCR-RFLPanalysis of nt3243 A to G mutation in MELAS patients. Total cell DNA from muscle or myoblasts (at indicated passage numbers) wasamplified by PCR with primers L316 and H330 and digested with HaeIII. Wild-type band is 140 bp long and mutant bands are 83 and 57bp. The positive control was obtained from a clone harboring the nt3243 mutation, the negative control from a clone from the wild-type.The elimination of mutant species occurred between P3 and P6 in MELAS2. (P5C is a cell clone from MELAS2 after five passages.) (d)PCR-DNA sequencing analysis of nt3243 mutation in MELAS patients. Total cell DNA from myoblasts (at indicated passage numbers)was amplified by PCR with the primer pair B1F1 and H388SQ; direct sequencing was performed by the dideoxy termination method (fmol,Promega). Open arrowheads indicate homoplasmy for the wild-type (nt A); closed arrowheads indicate heteroplasmy (nt A and nt G).


containing the nt3243 mutation are shown in Figure3c and d; estimates of the percentage of wild-type tomutant strains obtained by densitometric scan of thePCR-RFLP autoradiogram are shown in Figure 4.Here a more rapid elimination of the mutant specieswith in vitro proliferation is observed: in MELAS1,the percentage of mutant mtDNA was 45 in themuscle biopsy; this had decreased to 7% by P1.MELAS2 and MELAS3 muscle harbored higher lev-els of mutant mtDNA (83 and 86%, respectively); inMELAS2, this was only 4% by P3 and in MELAS3, itwas 14% by P7. This is confirmed by the sequencingresults, which show that MELAS1 is heteroplasmicfor the mutation in muscle and at P1, but not at P10or earlier; in MELAS2, the mutation is not apparentat P3, and in MELAS3, the cells are still heteroplas-mic at P7.

DISCUSSION

In Vitro Growth Characteristics. In all three patientgroups (MELAS, CPEO, and controls), there was anapparent trend for a decrease in CPDL with increas-ing passage numbers, in particular after the mid-stage of growth (Fig. 1a), with the MELAS patientgroup displaying the largest average decline (0.032),followed by the CPEO group (0.026) and the controlgroup (0.01). This observation implies that eitherthere is a genuine increase in generation time inculture with cell proliferation or that the increase

reflects selective cell attrition by apoptosis. There ishowever no evidence to suggest that cells in a givenculture will reach Hayflick20 point at widely differingtime points, indicating that the observed increase inCPDL is likely to reflect a true feature of in vitrogrowth. As the increase applied to the cytopathy aswell as the control cultures, it is likely that the samefactors are responsible in determining cell growth inboth groups; this in turn suggests that the Hayflickphenomenon is not the result of mt failure as previ-ously suggested by Goldstein,16 who attributed anincreased lactate production in old cells duringgrowth to a rise in energy demand and/or ineffi-ciency of OXPHOS. Failure of cell growth in a studywhere energy requirements were made independentof OXPHOS, via the use of pyruvate-supplementedmedia, supports the view that the OXPHOS factor isnot related to the Hayflick phenomenon.21,40

In addition, when CPDL was plotted against do-nor age for the control group, we observed a nega-tive correlation after establishment of the cell lines atabout P3 (Fig. 1b), suggesting that the regenerativecapacity of skeletal muscle may decrease with age, asa result of a reduction either in the number of sat-ellite cells present or in their differentiating abilityor both. Also, the nature and severity of the syn-drome appear to be significant in the determinationof CPDL as shown by MELAS3, who is 19 years old(CPDL of 0.1).

FIGURE 4. Estimation of the ratio of wild-type to mutant mtDNA in MELAS and CPEO patients with cell proliferation. Autoradiograms fromthe Southern blot (Fig. 3a) and the PCR-RFLP (Fig. 3c) were scanned in a phosphorimager to estimate the ratio of wild-type to mutantmtDNA for each MELAS and CPEO patient. The percentage of mutant mtDNA is plotted vs. passage number.


Respiratory Chain Activity. A study of the func-tional activity of the electron transport chain, in par-ticular the activity of complex I, complexes II–III,and complex IV with exhaustive in vitro cell division,revealed that two main processes are taking place inculture: (a) a progressive improvement in respirato-ry complex activity in cytopathy cell lines; and (b) asmall decline in respiratory chain activity in controlcell lines. The marked increase in complexes I andIV activity with serial passaging observed in the mtcytopathy group (Fig. 2) indicates that again, in thisin vitro model, RC failure does not contribute togrowth failure.

Similarly, the small overall decline in complex Iand IV activity observed in the control group sug-gests that this decline is unlikely to account for thegrowth exhaustion characteristics of this group.

mtDNA Genome. The phenotypic changes ob-served in the mitochondrial cytopathy myoblastswith cell growth, i.e., increase in RC activity and de-crease in some mitochondrial translation products,were associated with elimination of the mitochon-drial mutations from these cells until by mid to latepassage (P6 for MELAS2 and P14 for CPEO1), themutant genome was no longer detectable after PCRand therefore effectively eliminated (Figs. 3 and 4).This indicates that the process responsible for theimprovement in RC activity is elimination of mutantgenomes from the myoblasts; the process appears tobe more rapid for the point mutation in the MELASgroup than the common deletion in the CPEOgroup. It is of interest that in the case with a rela-tively high initial ratio of wild-type to mutant mtDNA(CPEO1), this ratio remains more stable with cellproliferation. We therefore have a situation wherecells harboring high levels of mutant mt genomesare eliminated with serial passaging.

Temporal analyses of the wild-type to mutantmtDNA expression in different models have yieldeddifferent results; in cybrid transformants for ex-ample, where a shift in a particular genotype hasbeen observed, whether the increase is directed athigher proportions of wild-type or mutant mtDNA isapparently determined by the nuclear genetic back-ground of the host cell line.13 Variant phenotypicexpression of mt genotypes was observed in skin fi-broblasts and Epstein–Barr virus (EBV) lymphocytesderived from the same Pearson patient; in the fibro-blasts carrying 60–80% deleted mtDNA, respiratorychain deficiencies were initially observed with pro-gressive recovery and gradual loss of the deleted ge-nomes as passage numbers increased; in the EBV-transformed lymphocytes carrying 60% deleted

genomes, the normal respiratory chain activity wasattributed to an increase in mtRNA translation effi-ciency.6,7 We believe however that skin is not an ap-propriate tissue for studying the expression of mtcytopathies, as it is well accepted that mtDNA muta-tions, especially deletions, are poorly expressed infibroblasts.33 It was also shown that in both skin fi-broblasts and skeletal muscle myoblasts obtainedfrom a Kearns–Sayre syndrome patient, only 2–4% ofdeleted mt genomes were found at early passages,compared to 50–60% in skeletal muscle.39

Mutation Elimination from the Mitochondrial GenomePool. The mechanism(s) underlying the rapidelimination of mutant genomes with improvementin cellular energetics in rapidly dividing cytopathymyoblast cultures are of great interest. Rapid segre-gation of mtDNA sequence variants has also beenobserved in other systems, i.e., heteroplasmic mice24

and Holstein cows,29 and the idea of rapid sortingout of mtDNA is generally well accepted.3 Thus, agenetic bottleneck for mtDNA in the female germ-line or early embryo was proposed whereby the ef-fective number of segregating mt units is reduced toa small number.1,19,27 It has recently been demon-strated that in heteroplasmic mice, this rapid segre-gation of mtDNA occurs early in oogenesis, duringexpansion of the oogonial population and prior todifferentiation of the primary oocyte population, bya process of random genetic drift24 rather than bynatural selection in the oocyte as initially sug-gested.30

In the context of pathogenic point mutations ofmtDNA, an important issue is whether there arepositive or negative selection pressures involved inthe transmission and segregation of mt genomes car-rying the mutations. The apparent rapid selection ofmt genotypes observed in different human maternallineages (1–2 generations) has been linked tomtDNA replication and to the possible physical‘‘packaging’’ of mtDNA genomes into clusters ornucleoids on the inner mt membrane.35,38 A longi-tudinal study of the nt3460 LHON mutation in afamily (heteroplasmic expression) suggests that seg-regation and transmission of mt genomes carryingthe LHON primary mutations are not subject to se-lection, but may rather reflect differences in the pro-portion of heteroplasmic mtDNA clusters and/ormitochondria within the germline.15

A similar process can be envisaged to be operat-ing in the rapidly dividing myoblasts in our study,whereby the more bioenergetically efficient wild-type mtDNA genomes/organelles are better physi-cally positioned than the mutant for mtDNA repli-


cation on the inner mt membrane and thereforepreferentially transmitted to the daughter cells. Wehave estimated the number of cell divisions takingplace before elimination of the mutant genomes tobe between 3 and 9 in the MELAS cell lines andbetween 11 and 26 for the CPEO cell lines. A similarmechanism may be operative in vivo; all cells gothrough a phase of division at some stage of theirdevelopment, and if similar mechanisms were to op-erate in vivo as in the in vitro cultures in our study,this cell division phase would involve a tendency to-ward reduction of the mutant mtDNA species,thereby contributing to the preservation of a pre-dominantly wild-type functional mt genome poolacross generations. Such a process does not precludeother selection events taking place at the oocytelevel.

Ms. J. Ojaimi is thanked for her help with the Southern transfers,Drs. Lawrie Austin and Robert Kapsa for their critical review of themanuscript, and Dr. Carolyn Sue (Dept. of Neurology, WestmeadHospital, New South Wales, Australia) for providing the clinicaldata on patient MELAS 1.

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Genotypic and phenotypic changes in exhaustively grown cell lines from mitochondrial cytopathy...

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