Mitochondrial DNA damage in lymphocytes: a role in immunosenescence?

Experimental Gerontology 37 (2002) 329±340

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Mitochondrial DNA damage in lymphocytes: a role in immunosenescence? Owen A. Ross a,b, Paul Hyland b, Martin D. Curran a,c,*, Brian P. McIlhatton a, Anders Wikby d, Boo Johansson e, Andrea Tompa d, Graham Pawelec f, Christopher R. Barnett b, Derek Middleton a,b,c, Yvonne A. Barnett b a

Northern Ireland Regional Histocompatibility and Immunogenetics Laboratory, Blood Transfusion Building, City Hospital, Belfast, Northern Ireland BT9 7TS, UK b School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland BT52 1SA, UK c School of Biology and Biochemistry, Queens University of Belfast, Belfast, Northern Ireland, UK d Department of Natural Science and Biomedicine, University College of Health Sciences, SE-551 11 JoÈnkoÈping, Sweden e Institute of Gerontology, University College of Health Sciences, SE-551 11 JoÈnkoÈping, Sweden f Tuebingen Ageing and Tumour Immunology Group, Section for Transplantation Immunology and Immunohaematology, Second Department of Internal Medicine, Medizinische Universitsklinik und Poliklinik, Otfried-Muller-Str. 10, D-72076 Tuebingen, Germany Received 1 June 2001; accepted 1 September 2001

Abstract An age-related increase of DNA damage/mutation has been previously reported in human lymphocytes. The high copy number and mutation rate make the mtDNA genome an ideal candidate for assessing damage and to act as a potential biomarker of ageing. In the present study, two assays were developed to evaluate the level of mtDNA 4977 and the accumulation of point mutations with age. A competitive polymerase chain reaction (PCR) methodology incorporating three primers was used to detect and quantify the levels of mtDNA 4977 and a novel heteroduplex reference strand conformational analysis (RSCA) technique was used to analyse the accumulation of point mutations. The assays were applied to an in vitro model of T cell ageing and ex vivo DNA samples from an elderly cohort of subjects and a younger control group. The mtDNA 4977 was detected in all the DNA samples examined but only a very low concentration was observed and no age-related increase or accumulation was observed. No accumulation of point mutations was identi®ed using RSCA within the T cell clones as they were aged or the ex vivo lymphocytes from the elderly cohort. A higher level of variation was observed within the ex vivo DNA samples, verifying the high resolution of RSCA and its ability to identify different mtDNA species, although no correlation with age was observed. The low level of mtDNA damage observed with respect to the ex vivo lymphocyte DNA samples within this study may be due in part to the high turnover of blood cells/mtDNA, which may inhibit the accumulation of genetically abnormal mtDNA that may play a role in immunosenescence. A similar explanation may also apply to the in vitro model of T cell ageing if the vast majority of the cells are replicating rather than entering senescence. q 2002 Elsevier Science Inc. All rights reserved. Keywords: Mitochondrial DNA; Damage; Lymphocyte; Immunosenescence

* Corresponding author. Address: Northern Ireland Regional Histocompatibility and Immunogenetics Laboratory, Blood Transfusion Building, City Hospital, Belfast, Northern Ireland BT9 7TS, UK. Tel.: 144-28-9026-3883; fax: 144-28-9026-3880. E-mail address: [email protected] (M.D. Curran). 0531-5565/02/$ - see front matter q 2002 Elsevier Science Inc. All rights reserved. PII: S 0531-556 5(01)00200-5

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1. Introduction An age-related increase in DNA damage/mutation has been reported in human lymphocytes (King et al., 1994, 1997; Barnett and King, 1995) and increased levels of oxidative DNA damage has also been reported using an in vitro model of T cell ageing (Barnett et al., 1999). Damage levels were observed to increase as the T cells aged in vitro under standard conditions (20% O2) and accumulation of such genetic abnormalities may result in cell cycle arrest (preventing T cell replication) or apoptosis if damage levels are critical (Barnett and Barnett, 1998; Hyland et al., 2000). Drouet and colleagues (1999), hypothesised that damage to the mitochondria could contribute to age-related immunode®ciency as mitochondria play a role in apoptosis, a major process in T cell death, and that damage may also result in insuf®cient ATP production, through oxidative phosphorylation (OXPHOS), for lymphocyte function (Shigenaga et al., 1994; Brenner et al., 1998; Drouet et al., 1999). The mitochondria are the major intracellular source of oxygen free radicals, usually referred to as reactive oxygen species (ROS), which cause cumulative damage to cellular constituents (DNA, RNA, proteins and lipids) that is postulated to result in ageing and eventual death. This process has been termed the mitochondrial theory of ageing (Brierley et al., 1997; Miquel, 1998; Barja, 2000; Brand, 2000; Cadenas and Davies, 2000; Kowald and Kirkwood, 2000; Melov, 2000; Rustin et al., 2000; Sastre et al., 2000; Berdanier and Everts, 2001; Salvioli et al., 2001). The mitochondria are distinct in that they contain their own DNA genome (mtDNA) that has been fully sequenced (Anderson et al., 1981). Each mitochondrion contains between 2±10 copies of mtDNA and due to its proximity to the main site of ROS production, the lack of protective histone proteins and an inef®cient DNA repair mechanism, the mtDNA undergoes mutation 5±10 times faster than nuclear DNA (nDNA). The established decline in mitochondrial bioenergetic function during human ageing in various tissues has been attributed to the occurrence and accumulation of mutations (deletions, base substitutions and frame-shifts) in the mtDNA (Brierley et al., 1998; Liu et al., 1998a; Graff et al., 1999; Michikawa et al., 1999; Fernandez-Moreno et al., 2000; Kopsidas

et al., 2000a; Wang et al., 2001). Deletions of mtDNA are the most common mutations associated with human diseases and the ageing process (Kovalenko et al., 1997; Cormio et al., 2000; Cottrell et al., 2000; Raha and Robinson, 2000). Deletions are epitomised by the `common deletion', a 4977bp mtDNA deletion (mtDNA 4977) that occurs, between two 13bp direct repeats, at nucleotide positions nt8470-13447. The 4977bp deleted region encodes for seven polypeptides essential for the enzyme complexes of the OXPHOS pathway; four for complex 1 (ND3, ND4, ND4L and ND5), one for complex IV (CO II), and two for complex V (ATP6 and ATP8) and ®ve tRNA genes (Porteous et al., 1998). The mtDNA 4977 has been reported to accumulate in a variety of human tissues with increasing age including heart, brain, lung, skeletal muscle (post-mitotic), skin and liver (non-post-mitotic) (Kao et al., 1997; Liu et al., 1998b; Bogliolo et al., 1999; Lu et al., 1999; Cormio et al., 2000; Kim et al., 2000). In the present study, two assays were designed to assess the level of damage that occurs within the mtDNA with age. The ®rst assay was designed to identify and quantify the occurrence of the 4977bp mtDNA ªcommon deletionº. This was performed by employing a competitive polymerase chain reaction (PCR) methodology incorporating a labelled forward primer and two unlabelled reverse primers, one of which was located within the region of the deletion and the other downstream from the deletion region. In the second assay, the level of mutation that occurs within the hypervariable D-loop was assessed using a novel heteroduplex methodology, reference strandmediated conformational analysis (RSCA) (Arguello et al., 1998). Results were obtained from a well characterised library of CD4 1 T cell clones that represent an in vitro model of T cell lifespan (Pawelec et al., 1997) and a sample of ex vivo lymphocytes from an aged population and a younger control population.

2. Materials and methods 2.1. Subjects Five (399-35, 399-37, 399-39, 400-23, 400-60) independently derived T cell clones were examined in this investigation (Pawelec, 1993). The £ 400

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series was obtained from a 26-year-old overtly healthy laboratory worker, and the £ 399 series was derived from a healthy 80-year-old donor conforming to the SENIEUR protocol for healthily aged individuals (Ligthart et al., 1984, 1990). The 50 elderly subjects were participants of the Swedish longitudinal NONA immune study (85±95 years old). A healthy sample of younger (30±60 years) women and men …n ˆ 15† was drawn among professionals working in the laboratories at the Ryhov Hospital in JoÈnkoÈping, Sweden, to act as controls. An additional 50 younger individuals (age-range of 19±45 years) were chosen at random from the DNA bank of normal healthy Caucasian individuals from the Northern Ireland population stored at the Northern Ireland Histocompatibility and Immunogenetics Laboratory (NIHIL) of Belfast City Hospital. 2.2. Culture of human peripheral blood derived CD4 1 T cell clones The ®ve human peripheral blood-derived T cell clones (expressing T cell receptors for antigen; TCR, CD3 1 , CD4 1 ), supplied by Professor Graham Pawelec (University of TuÈbingen Medical School, TuÈbingen, Germany, under the aegis of EUCAMBIS; Biomed 1 contract CT94-1209), were generated using a limiting dilution protocol as described earlier (Pawelec, 1993). Determination of the T cell lifespan and the culture conditions are as reported earlier (Hyland et al., 2000, 2001). The clones were grown in a serum-free medium, X-vivo 10 (BioWhittaker), and maintained at 37 8C in a 5% CO2, 95% air atmosphere in a 7-day cycle. At the end of each 7-day growth cycle, a new culture cycle was established with fresh medium, and aliquots were removed and cryopreserved in a medium made up of 10% DMSO, 20% foetal bovine serum (FBS) and 70% X-Vivo 10 and stored in liquid nitrogen prior to mtDNA damage analysis. 2.3. DNA extraction At speci®c monthly time points (23/3/00, 21/4/00, 11/5/00, 8/6/00, 13/7/00 and 4/8/00), during the in vitro lifespans of the ®ve T cell clones, aliquots were removed from storage and thawed, with the ®nal aliquot taken just prior to apoptosis of the cells. The salting out procedure (Miller et al., 1988)

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was employed to extract the DNA from the T cell clones (10 6 cells) and from whole blood in the case of the ex vivo lymphocyte DNA samples. All DNA concentrations in this study were established at 50 ng/ ml, an ample amount due to the high copy number of the mtDNA. 2.4. Detection and quanti®cation of mtDNA 4977 2.4.1. PCR ampli®cation of mtDNA for detection of the mtDNA 4977 A number of various techniques have been previously designed for the detection of the mtDNA 4977 e.g. long range PCR, serial dilutions and competitive PCR (Kovalenko et al., 1997; Porteous et al., 1998; Ahmed et al., 1999; Frippiat et al., 2000; Taylor et al., 2000; Mehmet et al., 2001). Described in this paper is an adaptation of the approach adopted by Sciacco et al., (1994). Our methodology is based on competitive PCR, employing three oligonucleotide primers to simultaneously amplify both wild-type mtDNA and mtDNA 4977. The forward primer CD1 (nt8340-8364) (5 0 GAG AAC CAA CAC CTC TTT ACA GTG A 3 0 ) was labelled at the 5 0 -end with the ¯ourochrome IRD800 (MWG Ltd. BIOTECH, Milton Keynes, UK). This forward primer was common to two reverse primers; CD2 (nt13614-13588) (5 0 TAT TCG AGT GCT ATA GGC GCT TGT CAG 3 0 ), which ampli®es outside of the deletion and SCD (nt8748-8720) (5 0 GGA TAC TAG TAT AAG AGA TCA GGT TCG TC 3 0 ), which ampli®es within the deletion region. The PCR product ampli®ed with the primer pair CD1 and SCD (wt-mtDNA) is 408bp compared to the PCR product ampli®ed to detect the presence of the deletion with the primer pair CD1 and CD2 (mtDNA 4977) which is 271bp. PCR ampli®cation was performed in 100 ml reaction volumes containing 67 mM Tris-HCl pH 8.8; 16 mM (NH4)2SO4; 1.5 mM MgCl2; 0.01% (w/v) Tween; 200 mM of each dNTP; 0.1 mM of each oligonucleotide primer; 2 units Taq polymerase (Bioline UK Ltd., London, UK); 0.05 mg DNA. The following cycling conditions were employed; samples were held at 96 8C for 5 min, followed by 30 cycles of 96 8C for 1 min, 60 8C for 1 min, and 72 8C for 1 min. After the ®nal cycle, the samples were held at 72 8C for 5 min, and then at 15 8C until removed from the PCR machine (Perkin±Elmer

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Fig. 1. Diagrammatic representation of the RSCA method. The FLR PCR product contains a ¯uorescent label on its sense strand shown by the star. The FLR is mixed with the PCR product of the 444bp D-loop region and the sense and antisense strands of the DNA amplicons are denatured. The strands are then allowed to re-anneal generating homoduplex, heteroduplex and single stranded FLR. The duplexes formed are then separated by PAGE and only those duplexes containing the FLR strand are detected with the laser. In this diagram, two mismatches are observed in the heteroduplex, which causes changes in the tertiary structure of the DNA strands, causing the migration pro®le to be different from that of the homoduplex FLR.

9600 Thermal Cycler). Successful ampli®cation was con®rmed using gel electrophoresis on a 1% agarose gel stained with ethidium bromide (0.5 mg/ml). Puri®ed PCR products (Qiagen QIAquick PCR puri®cation kit) from a number of individuals were sequenced on a 373A automated DNA sequencer (Curran et al., 1996) to con®rm the speci®city of the ampli®cation. 2.4.2. Analysis of ampli®cation products for quanti®cation of the mtDNA 4977 The T cell clones and the ex vivo DNA samples from an elderly cohort and a younger control group were analysed using a LI-COR 4200 automated sequencer (MWG Ltd. BIOTECH). Each sample (1 ml) was loaded onto a 5.5% non-denaturing polyacrylamide (Long Rangere Gel Solution; Flowgen, UK) gel and electrophoresis performed under constant conditions (1500 V, 25 mA, 25 W), while maintaining the temperature at 30 8C. The gel was 25 cm long, 0.25 mm thick and the loading wells were formed

with a 48 lane shark tooth comb. Gel image analysis was performed using Gene ImagIR (LI-COR Base ImagIR software CD version 2.3, LI-COR Inc. Biotechnology Division, Lincoln, NE 68504 USA) and quanti®cation was carried out using the RFLPscan Plus software (Scanalytics Inc., Billerica, MA 01821, USA). 2.5. RSCA analysis of point mutations in mtDNA Dloop 2.5.1. PCR ampli®cation of 444bp region of the Dloop The oligonucleotide primers employed for the ampli®cation of the 444bp region (nt15978-16422) of the D-loop were: D-loop II (nt15978-16001) (F) 5 0 CCA CCA TTA GCA CCC AAA GCT AAG 3 0 D-loop II (nt16422-16398) (R) 5 0 ATT GAT TTC ACG GAG GAT GGT GGT C 3 0

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PCR ampli®cation was performed as above with the following cycling conditions: samples were held at 96 8C for 5 min, followed by 35 cycles of 96 8C for 1 min, 56 8C for 30 s, and 72 8C for 1 min. After the ®nal cycle, the samples were held at 72 8C for 5 min. DNA sequence analysis was performed as above on a number of samples to con®rm the speci®city of the ampli®cation.

from a laboratory worker and was cloned into the pCR w 4 plasmid using the TOPO w Cloning Kit (Invitrogen BV, 9704 CH Groningen, Holland). A recombinant plasmid containing the 444bp region of the D-loop was sequenced on a 373A automated DNA sequencer (Curran et al., 1996) to establish its exact nucleotide sequence and was continually used as the source from which the FLR was synthesised.

2.5.2. Mutational analysis of 444bp region of the Dloop using RSCA To assess the level of mutational damage occurring within the mtDNA with age, a 444bp region of the hypervariable D-loop was analysed using RSCA, a novel heteroduplex methodology (Arguello et al., 1998). RSCA permits the sensitive detection of mismatches that occur between a ¯uorescent-labelled reference (FLR) strand and the complementary strand of an unlabelled sample PCR product. Fig. 1 provides a schematic diagram detailing the mechanism behind this technique. Essentially, a FLR PCR product is generated for the sequence of interest. The ¯uorescent label is restricted to one of the DNA strands of the FLR PCR product as only one of the two PCR primers contains a 5 0 ¯uorescent label. The FLR is then mixed with the PCR product for the same region (unlabelled) from a different sample to be tested. A process of denaturation followed by annealing is allowed to take place to facilitate hybridisation of the complementary sense and antisense strands. Annealing can also occur between the sense and antisense strands of the different DNA species present in the mixture, forming heteroduplexes (Fig. 1). The duplexes formed are separated by non-denaturing polyacrylamide gel electrophoresis (PAGE) using an automated sequencer and only those duplexes possessing a ¯uorescent label are detected by the laser. The nucleotide mismatches that occur between the DNA strand of the FLR and the DNA strand of the sample cause loop outs in the tertiary structure of the heteroduplex, which retards its migration, relative to the homoduplex, during electrophoresis, producing highly reproducible migration patterns (RSCA pro®les).

2.5.4. Reference strand conformational analysis (RSCA) Brie¯y, the FLR of known sequence was ampli®ed from the recombinant plasmid for the 444bp D-loop region using the D-loop II primers as above, except the forward primer contained a 5 0 ¯uorescent label (¯ourochrome IRD800, MWG Ltd. BIOTECH, Milton Keynes, UK). The FLR (1 ml) was then mixed in a 1:3 ratio with the unlabelled ampli®ed PCR product (444bp D-loop region) derived from each sample for RSCA analysis. A process of denaturation and re-annealing was facilitated via the following conditions; 96 8C for 4 min, 50 8C for 10 min, followed by 15 8C for 20 min. The samples were then held at 4 8C until they were removed from the PCR machine (Perkin-Elmer 9600 Thermal Cycler). The FLR-sample mixture (4 ml) was mixed with an equal volume of Ficoll loading buffer (4 ml; 15% Ficoll, 0.25% bromophenol blue) and 1 ml was loaded onto a 5.5% non-denaturing polyacrylamide gel (Long Rangere Gel solution; Flowgen, UK) and electrophoresis was performed using a LI-COR 4200 automated sequencer under constant conditions (1500 V, 25 mA, 25 W), while maintaining the temperature at 30 8C. The gels were 66 cm long, 0.25 mm thick and the loading wells were formed with a 48 lane shark tooth comb. Each FLR duplex combination was visualised and analysed after electrophoresis using RFLPscan Plus software (Scanalytics Inc., Billerica, MA 01821, USA).

2.5.3. Cloning and DNA sequencing of FLR To ensure that the reference chosen contained only one mtDNA species of known sequence, the reference sequence was PCR ampli®ed from the DNA extracted

3. Results 3.1. Detection and quanti®cation of mtDNA 4977 A competitive PCR methodology incorporating three primers, to simultaneously amplify both wtmtDNA and mtDNA 4977 was employed for the

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Fig. 2. A representative PAGE image for the detection and quanti®cation of mtDNA 4977. The gel image distinguishes between wt-mtDNA (408bp) and mtDNA 4977 (271bp) based on size. The lanes M contain molecular weight marker (microSTEP-13a, IRD800 labelled, MWGBIOTECH). Lanes 1±6 and 7±12 display the results obtained for one T cell clone (399-35) following PCR ampli®cation over 30 and 35 cycles, respectively. Each lane refers to a sample removed at monthly intervals during the clones lifetime with the ®nal sample (lane 6 and 12) taken just before apoptosis of the cells. Lanes 1C±8C represent the results obtained for eight of the ex vivo DNA younger subjects and lanes 1A±8A correspond to a selection of eight of the ex vivo DNA samples from the cohort of elderly individuals. No accumulation of mtDNA 4977 was observed during the lifespan of any of the T cell clones and as represented in this ®gure the concentration of mtDNA 4977 in the ex vivo DNA samples only varied between individual samples showing no age-related increase or accumulation.

detection and quanti®cation of mtDNA 4977. When PCR ampli®cation was performed over a range of cycles (20, 25, 28, 30 and 35), the least number of cycles that would permit detection of mtDNA 4977 in all the samples was 30. However, quanti®cation analysis of the PCR products derived from each of the different cycle numbers revealed that the smaller mtDNA 4977 product was being preferentially ampli®ed in the later cycles and therefore, both PCR products (wt-mtDNA and mtDNA 4977) were no longer accumulating in an exponential fashion. This is particularly evident when 30 cycles is compared to 35 cycles over the lifespan of T cell clone (399-35) in Fig. 2. As our quantitative analysis predicts the exponential ampli®cation phase ends between 20±25 cycles, this would be the optimum time to quantify the level of mtDNA 4977 in comparison to the wtmtDNA; however, because the mtDNA 4977 is at such a low concentration (estimated at less than 0.01% of the wt-mtDNA), it is undetectable at this stage of the ampli®cation. To establish whether there is an increase in the concentration of mtDNA 4977 with age, all ampli®cations were performed over thirty cycles, although this was outside of the exponential ampli®cation phase all the sample DNA concentra-

tions were standardised at 50 ng/ml to remove any variation between samples due to the preferential ampli®cation. When PAGE was carried out and quanti®cation was performed on the T cell clones and the ex vivo DNA samples of an elderly cohort of subjects …n ˆ 50† and a younger control group …n ˆ 65†; the deletion was observed in all the samples. The concentration of mtDNA 4977 detected was low and varied with respect to each individual but not in an age-related manner. This absence of an age-related accumulation is clearly shown in lanes 1±6 of the representative gel image (Fig. 2) displaying the results obtained for the lifespan of one of the T cell clones (399-35) ampli®ed over thirty cycles i.e. the intensity of the mtDNA 4977 remains constant throughout its ageing process. These results were also con®rmed at 35 cycles (Fig. 2), although the intensity was greatly increased due to the preferential ampli®cation. In the case of the ex vivo DNA samples, the representative ®gure shows eight younger individuals compared to eight elderly subjects and there is clearly no age-related accumulation of mtDNA 4977. However, there are two individuals one from each age group (lanes 5C and 2A) containing a higher concentration of the mtDNA 4977

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Fig. 3. A representative image of the RSCA Analysis of the 444bp D-loop region. The lanes M contain molecular weight marker (microSTEP13a, IRD800 labelled, MWG-BIOTECH). Lane R contains only the reference homoduplex. Lanes 1±6 display the results obtained for one T cell clone from the £ 399 series (399-35), displaying RSCA pro®le 1. Lanes 7±12 display the results obtained for one T cell clone from the £ 400 series (400-23), displaying RSCA pro®le 2. Each lane refers to a sample removed at monthly intervals during the clones lifetime with the ®nal sample (lane 6 and 12) taken just prior to apoptosis of the cells. Lanes 1C±8C represent the results obtained for eight of the ex vivo DNA younger subjects and lanes 1A±8A correspond to a selection of eight of the ex vivo DNA samples from the cohort of elderly individuals. Lane 4C is displaying polyplasmy (three distinct heteroduplex bands are clearly visible).

compared to the others, highlighting the variation between individuals. 3.2. Mutational analysis of mtDNA D-loop using RSCA A 444bp region of the mtDNA D-loop (nt1597816422) was analysed using the highly sensitive heteroduplex methodology RSCA, to act as a marker for DNA damage within the cell. RSCA was performed on the library of T cell clones to assess the accumulation of point mutations within the Dloop region, as they were aged. Within each T cell clone, one retarded heteroduplex (RSCA pro®le) was observed distinct from the homoduplex through its unique migration distance during PAGE (Fig. 3). Analysis of the ®ve independent T cell clones identi®ed two distinct RSCA pro®les, one of which was unique to the three T cell clones of the £ 399 series and the other to the £ 400 series, indicating two different mtDNA species (pro®les 1 and 2 in Fig. 3). One should highlight that the homoduplex band may correspond to a second mtDNA species identical in sequence to the reference within each T cell clone. No additional heteroduplex bands were observed as the ®ve independent T cell clones underwent in vitro ageing, and this is clearly visible in Fig. 3, which displays the results obtained for one T cell clone

from the £ 399 series (lanes 1±6) and one from the £ 400 series (lanes 7±12). This data therefore indicates the absence of an accumulation of point mutations within the hypervariable D-loop region of the mtDNA within these T cell clones as they age. The D-loop region of the ex vivo DNA samples displayed a much higher degree of variation (shown in Fig. 3). A total of 16 different RSCA pro®les were observed across the total number of samples …n ˆ 115† examined, eight of which are shown in the representative ®gure (Fig. 3). The majority of samples in both the elderly cohort of subjects (82%) and the younger individuals (86%) contained only one distinct RSCA pro®le and the homoduplex band, indicating one predominant mtDNA species other than the reference (homoduplex band). The two RSCA pro®les observed in the T cell clones (Fig. 3) were the most common mtDNA species found in the ex vivo DNA samples. When we examined the distribution of the various types/RSCA pro®les between the two age groups, no statistically signi®cant frequency differences were observed, although this was not surprising considering the high number of variables. A level of polyplasmy was observed in the ex vivo DNA samples (see lane 4C in Fig. 3) with the maximum number of mtDNA species in any one sample totalling four. However, there was no signi®cant agerelated increase or decrease in polyplasmy (multiple

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heteroduplexes) between the elderly cohort of subjects (n ˆ 50; 14%) and the younger group (n ˆ 65; 18%), indicating an absence of an accumulation of mutated mtDNA species with age. 4. Discussion There is still relatively little known about modi®cations in the mitochondria of the immune system cells with ageing (Drouet et al., 1999). As the mitochondria play a central role in apoptosis and in ATP production, age-associated defects in mitochondrial function are postulated to have an importance in T cell function and therefore immunosenescence. It is now established that during human ageing, there is a decline in mitochondrial bioenergetic function and this has been attributed to the accumulation of genetic abnormalities within the mtDNA (Porteous et al., 1998; Kopsidas et al., 2000a). The objective of the present study was to examine the hypothesis that mtDNA deletions and point mutations accumulate in the lymphocytes with age. To assess the level of mtDNA deletion accumulation, a competitive PCR methodology was employed to detect the age-associated common deletion, mtDNA 4977. As the mtDNA 4977 is associated with a variety of pathological and physiological defects, it may be exploited as a sensitive and early marker of generalised mitochondrial impairment. In order to detect the mtDNA 4977 with our competitive PCR system, it was necessary to perform the ampli®cation outside the exponential phase (20±25 cycles) impairing the quanti®cation of the exact ratio of mtDNA 4977/ wt-mtDNA in each sample. Our analysis did not reveal an increase in mtDNA 4977 with age, in either the T cell clones or the ex vivo lymphocyte DNA samples, the mtDNA 4977 band intensity remained broadly constant throughout (Fig. 2), with the exception of a few individuals in the ex vivo lymphocyte DNA samples, displaying a higher concentration that re¯ects individual variability. Since our DNA samples were all at the same concentration (minimising the affect of variable preferential ampli®cation between the samples), any age-related increase should have been detected. Preferential ampli®cation of deleted mtDNA species is a major concern especially when using

long range PCR and competitive PCR involving different primer sets (Kopsidas et al., 2000b; Mehmet et al., 2001). Mehmet et al. (2001) recently found that the serial dilution method can over-estimate the levels of mtDNA 4977 in samples by up to 1000-fold, due to its preferential ampli®cation. The fragment size has also been reported to affect the ampli®cation ef®ciency, as there is approximately a 50% drop in the detection of mtDNA 4977 when an eight hundred base fragment was used in PCR ampli®cation rather than a four hundred base pair product (Von Wurmb et al., 1998). In the present study, relatively small fragments (wt-mtDNA 408bp and mtDNA 4977 271bp), ensuring a low size difference between products (137bp), were selected in our PCR design in order to minimise the level of this effect. Nevertheless, preferential ampli®cation was observed in this study, highlighting that accurate quanti®cation of the ratio of mtDNA 4977/wt-mtDNA can only be performed during the exponential PCR phase (,25 cycles), which was below our detection limit. The results of the present study con®rm previous ®ndings that indicate that the average mtDNA 4977 concentration is very low in normal (non-diseased) tissues and cells and therefore it is very important for an assay to be both quantitative and accurate at a very extreme mtDNA 4977/wt-mtDNA ratio range (0.1±0.0001%) (Mehmet et al., 2001). Recently, Meissner et al. (2000) reported a real-time PCR procedure using the ABI Prism 7700 sequence detection system with ¯uorescent probes, which is capable of detecting and quantifying the level of mtDNA 4977 in the blood of healthy individuals. A substantially lower mtDNA 4977 concentration in blood is reported than in post-mitotic tissues and in agreement with our results no age-related accumulation was observed (Meissner et al., 2000). This, together with our data raises the possibility that levels of the mtDNA 4977 may have been over estimated in earlier studies that employed high PCR cycle numbers (.25 cycles). A number of earlier investigations have failed to detect the deleted form in the blood of healthy individuals, although increased concentrations of mtDNA 4977 have been consistently reported in the blood of diseased (e.g. Kearns-Sayre syndrome, Pearson's pancreas syndrome and mitochondrial myopathies) patients (Biagini et al., 1998; Von Wurmb et al., 1998). Interestingly, we detected the presence of

O.A. Ross et al. / Experimental Gerontology 37 (2002) 329±340 4977

mtDNA in all the individuals examined (T cell clones and ex vivo blood samples), although at an extremely low level, suggesting that PCR ampli®cation conditions are critical for its detection. The low occurrence of mtDNA 4977 during in vitro T cell ageing and in the ex vivo lymphocyte DNA samples, would indicate that there is no signi®cant accumulation of this genetic abnormality in the cells of the immune system with age. Point mutations within the mtDNA are postulated to accumulate with age (Michikawa et al., 1999; Wang et al., 2001). To test this hypothesis, it was decided to exploit the hypervariable nature of the Dloop region of the mtDNA. The 1122bp D-loop of the mtDNA is a non-coding region that is located between nt16,024-576 and is known to contain a number of polymorphic variants. Mutational analysis of a 444bp section of the D-loop (nt15978-16422) was performed using a novel heteroduplex methodology, RSCA, which identi®es sequence variations through differing band migration patterns obtained via nondenaturing PAGE (Arguello et al., 1998). The assay was applied to the in vitro model of T cell ageing and the ex vivo DNA samples of an elderly cohort of subjects and a younger control group. As with the mtDNA 4977 assay, no accumulation of damaged mtDNA was observed within the T cell clones, as they aged (Fig. 3), the RSCA pro®les observed did not alter during their lifespan. The high resolution of RSCA identi®ed a total of 16 different mtDNA species (RSCA pro®les) within the ex vivo DNA samples, although the majority of the samples contained only one distinct mtDNA species other than the reference. On examination of the 16 various RSCA pro®les observed within the two age groups, no statistically signi®cant associations were revealed. A level of polyplasmy was however observed in both age groups although surprisingly, no difference with age was observed. From these results, one can conclude that a variety of mtDNA species exist within cells of the immune system in vivo but this more than likely re¯ects the polymorphic nature of the mtDNA genome (Ross et al., 2001) rather than any accumulation of mtDNA damage with age. Certainly, the similar polyplasmic frequencies observed in both age groups does not support the hypothesis that genetic abnormalities of the mtDNA play a signi®cant role in the dysfunction of the immune system with age.

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The low level of mtDNA damage with age that was observed in this study would suggest that the mitochondrion may not be as prone to DNA damage as previously believed. A major hypothesis in the mitochondrial theory of ageing is the cumulative damage to the mtDNA by ROS. Recently, however, it has been reported that mitochondrial endogenous oxidative damage may have been over estimated in the past (Anson et al., 2000) and although mtDNA does not encode DNA repair proteins (Bohr and Anson, 1999), repair of damaged mtDNA does take place, compensating for oxidative damage (Bohr and Anson, 1999; Bohr and Dianov, 1999). Antioxidant defences and DNA repair mechanisms have been shown to affect the level of DNA damage and age-related changes in these parameters have been reported in lymphocytes (Mendoza-Nunez et al., 1999; Doria and Frasca, 2001). The absence of an age-related increase in the level of mtDNA damage may in part be due to the high turnover rate of blood cells, which does not permit the damaged mtDNA to accumulate in any signi®cant number (Meissner et al., 2000). It is also worth noting that mtDNA is actively turned over in both mitotic and post-mitotic tissue every 7±31 days, depending on the cell type and tissue, therefore ridding itself of many of the mutant forms (Kopsidas et al., 2000a). Certainly, the above factors may address why our analysis observed an absence of mtDNA damage within the ex vivo lymphocyte samples. With respect to the T cell clone model of ageing, it is somewhat more dif®cult to explain the lack of mtDNA damage observed in the aged cells, just prior to apoptosis. When one considers what is known about the growth kinetics of the T cell ageing model, one cannot dismiss the possibility that survival of the ®ttest is occurring in this model and therefore, the arguments raised above for the blood cells may well apply. This is particularly relevant as the fraction of dividing versus quiescent/senescent cells during each culture cycle is currently unknown for each of the individual T cell clones and may therefore have had a bearing on our investigation. Only when the growth kinetics of the in vitro T cell clone model of ageing is fully elucidated, can we really conclude whether our observed absence of mtDNA damage in the T cell clones is due to a preponderance of rapidly replicating cells or re¯ects a true absence of damage in senescent cells.

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