Mutagenesis vol. 19 no. 2 pp. 99-104,
March 2004
© 2004 UK Environmental Mutagen Society/Oxford University Press
Individual susceptibility to DNA telomerase inhibitors: a study on the chromosome instability induced by 3'-azido-3'-deoxythymidine in lymphocytes of elderly twins
Department of Public Health and Cell Biology, University of Rome ‘Tor Vergata’, via Montpellier 1, I-00133 Rome, Italy and 1Human Biology Centre, University Institute of Movement Science (IUSM), Rome, Italy
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The activation of telomerase in phytohemagglutinin-stimulated peripheral lymphocytes is thought to play a role in telomere maintenance and DNA repair. Considering the importance of this enzyme in both cancer and senescence, we studied the effects of the telomerase inhibitor 3'-azido-3'-deoxythymidine on the frequency of chromosomal aberrations and micronuclei induced in peripheral blood lymphocytes (PBL) of elderly monozygotic and dizygotic twins, evaluated with respect to the genotoxic effects induced in unrelated young subjects. Our results show that the cytogenetic damage induced by 3'-azido-3'-deoxythymidine in human PBL was mainly regulated by genetic factors and allowed the identification of hypersensitive subjects. Ageing, which did not modify the individual susceptibility to 3'-azido-3'-deoxythymidine induction of chromosome aberrations and micronuclei, nevertheless determined an overall increase in nuclear damage.
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Introduction |
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In vivo and in vitro cellular longevity is determined by a multiplicity of genetic factors which, interacting with the environment in different and not completely known ways, lead to senescence. Among the cellular and molecular markers of senescence, the analysis of chromosome instability and telomere erosion could represent old and new approaches to the same biological determinant.
As predicted by Jacobs as early as 1961 (Jacobs et al., 1961
), an increase in chromosomal instability is a cytogenetic feature of chronological ageing in humans. Association between chronological ageing and aneuploidy in cultured human lymphocytes has been described in different reports (Fitzgerald and McEwan, 1977; Galloway and Buckton, 1977) and, in addition to non-disjunction, chromosome elimination by micronuclei (MN) formation could account for age-related aneuploidy (Fenech and Morley, 1985; Bukvic et al., 2001). Moreover, ageing also seems to be related to an enhancement of structural chromosome aberrations (CA), i.e. unstable aberrations, stable translocations and symmetrical exchanges, the frequency of which seem to increase linearly with age (Ramsey et al., 1995; Tucker and Moore II, 1996).
In recent years, the study of telomeres, the eukaryotic chromosome ends composed of specific DNA repeat sequences and associated proteins (see Blackburn, 2000, for a review), has strongly pointed to a possible role of telomere length and maintenance in senescence and some age-related disorders, including increasing incidence of cancer, atherosclerosis, osteoporosis, diminished skin elasticity and weight loss. (Aviv and Aviv, 1998). Indeed, studies on telomerase-deficient mice have demonstrated a critical role for telomere length in the overall fitness, reserve and well-being of ageing organisms (Rudolph et al., 1999). Although it is hard to directly correlate telomere shortening with organismal ageing, there is a close relationship between replicative senescence and telomere length in human cells (Harley et al., 1990; Vaziri et al., 1994; Johnson et al., 1998) and recently a clear correlation has been demonstrated between structural and numerical aberrations in chromosomes and telomere erosion and/or telomerase activity both in vitro (Filatov et al., 1998) and in vivo (Samper et al., 2001) models. Actually, telomerase, the cellular reverse transcriptase that elongates telomeres de novo using an RNA molecule component as template (see Nugent and Lundblad, 1998, for a review), shows low or undetectable activity in many somatic tissues from humans, but is up-regulated in the majority of human tumours (Shay and Bacchetti, 1997) and in germ cells, activated leukocytes and stem cells (Harley et al., 1994). On this basis, it has been hypothesized that telomere length maintenance by telomerase could be crucial for high turnover organs, such as the skin, lymphatic system and gastrointestinal tract, whose specific alterations in ageing could be the effect of accelerated loss of telomere repeats (Campisi, 1997).
Thus, since the degree of telomerase efficiency in these tissues could be a determinant of ageing-related chromosomal instability, we have investigated the role of ageing and genetic factors in the chromosomal instability induced by a telomerase inhibitor, the nucleoside analogue 3'-azido-3'-deoxythymidine [zivudine, azidothymidine (AZT)] (Yegorov et al., 1996; Gomez et al., 1998). Phytohaemagglutinin (PHA)-stimulated peripheral lymphocytes from elderly monozygotic and dizygotic twins, and from unrelated young controls, were analysed for the frequency of CA and MN induced in vitro by AZT. The results show that: (i) ageing correlates with a higher susceptibility to nuclear damage induced by this telomerase inhibitor; (ii) some individuals have a specific high sensitivity to the clastogenic effect of the telomerase inhibitor; (iii) the individual susceptibility is highly genetically determined, and maintained during ageing, over the environmental factors.
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Materials and methods |
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Subjects
A total of 58 healthy male subjects were considered in this study, including 10 monozygotic (MZ) and 12 dizygotic (DZ) pairs of elderly twins (mean age 71.0 ± 2.6), randomly selected from the Rome Twin Register (Casini et al., 2002
). A genotypic analysis using four microsatellite loci (D1S80, D3S1358, vWA and FGA) and methods previously described (Torrente et al., 1998) confirmed twin zygosity. A group of 14 unrelated healthy younger males (mean age 34 ± 5.2) was analysed as a control group to verify age and genetic effects on clastogenic sensitivity to AZT. The blood samples from MZ, DZ and control subjects were randomly collected during the experimental period.
Cell culture and exposure
After informed consent, heparinized blood samples were collected from the 58 subjects and, for each subject four short-term cultures of peripheral lymphocytes were settled by adding 0.5 ml of whole blood to 10 ml of culture medium (RPMI 1640) supplemented with foetal bovine serum (10%) and PHA (2%). At 48 h after the start, 50 µg/ml AZT (Sigma) (a stock solution of 10 mg/ml in physiological solution) was added to two cultures for the last 24 h, while to the other cultures physiological solution alone was added. The AZT dose was chosen on the basis of a preliminary experiment performed with the drug at final concentrations of 50 and 100 µg/ml. Cultures were then split for CA and MN analyses and maintained at 37°C for a total of 72 h in a humidified incubator.
Chromosomal aberrations and micronuclei analyses
To obtain metaphase preparations, cultures for CA analysis were supplemented with colcemid (0.2 µg/ml) for the last 2 h and then processed by standard methods. For each treatment, 100 Giemsa stained metaphases were scored on coded slides. Mitotic index value, analysed to verify individual differences in PHA mitogenic activity, was determined as the percentage of metaphases/1000 observed nuclei. Both damaged metaphases and CA were considered. Gaps were not included in the analysis. Dicentrics, rings, acentric fragments and asymmetrical translocations were recorded separately. At the same time as AZT addition, cells for MN analysis were arrested in cytodieresis with cytochalasin B (6 µg/ml) (Sigma) and harvested at 72 h according to the CBMN standard protocol. MN count was evaluated by scoring 1000 binucleated cells/sample on coded slides. In the control group, MN analysis was performed on 9 of 14 subjects.
Statistical analysis
When possible, a t-test was used to compare mean frequencies. The Mann–Whitney rank sum U-test and Spearman rank correlation test were chosen for a group-wide evaluation of the individual data that did not follow a normal distribution. To avoid the influence of the same genetic factors in the assessment of the role of ageing in damage induction, we formed a ‘reference group’ from the elderly twin sample. This group, consisting of 22 unrelated elderly subjects, was made by choosing at random only one subject for each twin pair. For the correlation analysis, 20 unrelated couples were formed by randomly matching one monozygotic twin with one dizygotic twin.
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AZT-induced breakage shows individual susceptibility
Based on preliminary experiments performed on lymphocytes from three healthy young donors exposed to AZT for different lengths of time and at different concentrations (50 and 100 µg/ml), we chose to apply treatment with 50 µg/ml of AZT for the last 24 h of culture in a study on lymphocytes from aged and control groups. As shown in Figure 1, this treatment with AZT was sufficient to induce a statistical enhancement of nuclear damage, with a minor effect on proliferation index values, heavily compromised at higher drug concentrations. Independently of age, AZT treatment of lymphocytes induced a significant increase in the mean values of aberrant cells (AC), CA and MN in all groups (Table I). Differences in the mean values of CA can be noted in the aged groups in relation to the subdivision into mono- or dizygote, and this result was due to one MZ pair (S7A and S7B) showing the highest CA frequencies among all the 58 subjects analysed (91 and 209%, respectively). The proportion of aberrations per aberrant cell did not show significantly different values (1.4–1.6, P > 0.05) between groups, with a predominant induction of chromatid-type aberrations (Figure 2), but within each group the average frequency of CA showed a high standard deviation, due mainly to the presence of individuals who showed a specific hypersensitivity to AZT-induced nuclear damage (Table II). The subjects showing a CA frequency higher than the mean + SD of the group were designated CA-hypersensitive subjects and represented about 20 and 30% of the individuals in the control and elderly groups, respectively. For the MN frequencies, the SD value did not identify specifically high responses and, actually, no significant correlation (P > 0.05) could be detected between individual susceptibility towards CA and MN induced by AZT.
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AZT induces increased nuclear damage with ageing and its effect is highly determined by genetic factors
Ageing was associated with a specific enhancement of nuclear damage induced by AZT in peripheral lymphocytes, with higher frequencies in the elderly reference group than in controls of aberrant cells (21.4 ± 2.7 versus 14.8 ± 1.8), CA (39.4 ± 8.8 versus 22.5 ± 4.1) and MN (26.7 ± 2.9 versus 12.4 ± 3.2) (P < 0.05) (Figure 3A). Since spontaneous levels of CA and MN were statistically higher in the elderly versus controls, as already described (Barale et al., 1998
), the net increment due to AZT treatment was also considered, leading to a similar result (Figure 3B). Moreover, a statistical comparison of mitotic index values did not show differences (P > 0.05) between groups, neither considering young (2.44 ± 0.79) versus elderly (2.32 ± 1.24) subjects nor considering normal (2.28 ± 1.30) versus hypersensitive (2.51 ± 0.90) subjects, thus eliminating the possibility that AZT sensitivity could be related to differential responsiveness of lymphocytes to PHA mitogenic activity. To further verify that ageing enhanced the overall individual susceptibility to AZT-induced CA, rather than the expression of a specific hypersensitivity to AZT, we compared young and aged individuals within both the normal and the hypersensitive groups. Figure 4 clearly shows that ageing enhanced AZT-induced aberrations in both groups.
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Our results also show that genetic factors strongly influence individual sensitivity towards this telomerase inhibitor. Actually, among monozygote twins both CA (r = 0.92, P < 0.0001) and MN (r = 0.80, P < 0.01) frequencies show a high degree of correlation, absent among dizygote twins (r = 0.60, P > 0.05 for CA; r = 0.27, P > 0.05 for MN) and unrelated subjects (r = 0.38, P > 0.05 for CA; r = – 0.07, P > 0.05 for MN) (Figure 5). The presence of a high concordance for the CA-hypersensitive phenotype, observed also in dizygotic twins, strongly influenced the correlation coefficients for CA. When hypersensitive twins were excluded from the analysis, the correlation coefficients were appreciably lower for both the dizygotic (r = 0.10) and monozygotic (r = 0.79) groups, but still maintaining statistical significance (P < 0.05) in the latter case.
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Discussion |
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The nucleoside analogue AZT, widely used in the treatment of HIV-infected patients and in the prevention of maternal viral transmission of HIV during pregnancy (IARC, 2000
; Lyall et al., 2001), is active against retroviral replication by inhibiting retroviral reverse transcriptase (Furman et al., 1986). In the cell, this drug is converted into the active triphosphate form and is incorporated instead of the respective deoxynucleoside 5'-triphosphate (Furman et al., 1986).
Our study shows that lymphocyte sensitivity to the genotoxic activity of AZT, already proved through different end-points (Gonzales-Cid and Larripa, 1994; Meng et al., 2000), increases with ageing, with an individual variability largely determined by genetic factors. Although individual variability to AZT sensitivity may be related to polymorphisms of enzymes known to mediate the cellular activity of AZT, such as the major phase II drug metabolizing enzymes UDP-glucuronosyltransferase (Resetar and Spector, 1989; Bock, 2003) or antioxidant enzymes (Prochaska et al., 1993; Magnani et al., 1997), the higher genotoxic effect of AZT in lymphocytes from elderly subjects suggested an involvement of ageing-related processes. Since AZT incorporation can interfere with different processes related to DNA metabolism, i.e. chain elongation during DNA synthesis (Furman et al., 1986) or exonucleolytic repair activity (Harrington et al., 1993), the clastogenicity of AZT may arise from the inhibition of cellular processes related both to chromosome maintenance and repair in lymphocytes.
Olivero and Poirier (1993) showed that AZT is also selectively incorporated into most telomeres, acting as a telomerase inhibitor. Long-term AZT treatment induces shortening of the telomeric sequences of HeLa cells (Gomez et al., 1998), decreases the telomere length in immortalized human cell lines (Strahl and Blackburn, 1996), inhibits cell proliferation with senescent-like phenotype induction in cultured mouse fibroblasts (Yegorov et al., 1996) and shortens the telomeres of mice exposed in utero (Olivero et al., 1997). It has been suggested that telomerase activity in PHA-stimulated lymphocytes, up-regulated when cells progress from G0 to S phase, may play an important role in the repeated clonal expansion of lymphocytes (Yamada et al., 1996). Indeed, apparently stable telomere lengths have been detected in the initial culture period of T cells, coincident with the highest levels of telomerase activity, suggesting that telomerase maintains telomere length in these cells (Bodnar et al., 1996). Moreover, UV-, 
- and X-irradiation induce telomerase activity in cells of different origins (Leteurtre et al., 1997; Neuhof et al., 2001), while radiosensitive murine and human cells show telomere length abnormalities (Khanna et al., 2001; McIlrath et al., 2001), with a possible role for telomeres and telomerase in the DNA damage response (Kishi and Lu, 2002).
Thus, the type and extent of nuclear damage induced by the telomerase inhibitor AZT in peripheral lymphocytes could be determined by telomerase involvement both in chromosome ends maintenance and in repair of DNA damage (i.e. DNA double-strand breaks) leading to chromosomal breakage. Our results may reflect the modulation of genetic factors and ageing in these phenomena. Actually, our findings are fully in agreement with the study of Kosciolek and Rowley (1998) on the heritability of telomerase activity. In their study, performed on a sample of 23 twins of different ages, the authors found that the degree of telomerase activity inducible in lymphocytes is determined mainly by genetic rather than by environmental factors, with a coefficient of heritability of 0.814. From their data it also appears that 
13% of the subjects showed specifically high telomerase activity, which can partially explain the proportion of subjects hypersensitive to AZT induction of CA that we found in our study. With respect to MN, we did not find specific hypersensitivity to AZT induction. This difference, besides technical variations, could be related to the fact that although chromosomal breakage results in MN formation, a high proportion of MN harbour whole chromosomes (Fenech and Morley, 1989). Actually, the majority of AZT-induced CA included chromatid and isochromatid breaks and exchange, related to mechanisms other than mitotic non-disjunction or micronucleation (Catalan et al., 1995; Hagmar et al., 1998). Moreover, the CA hypersensitivity was often paralleled by a decrease in the proportion of binucleated/mononucleated cells (data not shown), probably caused by loss of highly damaged cells.
Our results also show that ageing does not modify the degree of individual susceptibility to AZT clastogenic activity, the percentage of hypersensitive individuals being the same in the two age groups, but enhances the overall effect of the telomerase inhibitor on CA and MN induction. The finding that ageing does not significantly alter the capacity for telomerase induction (Son et al., 2000) could explain this result, and the differences found in the aged twins for the total CA and MN frequency could reflect a general impairment of the cellular response to nuclear damage (Burkle, 2002). In addition, our results may contribute to improve the utilization of AZT in both HIV and, more recently, tumour therapy. Some clinical studies suggest that the levels of incorporation of AZT into DNA observed in human cells from HIV-infected mothers and infants treated with AZT is sufficiently high to be mutagenic (Olivero et al., 2000). The same study suggests the existence of interindividual differences (perhaps genetic) affecting the levels of DNA incorporation of AZT and, thus, the risk for mutations. Transplacental exposure to a single agent or a combination of drug regimes may constitute a potential cancer risk for HIV-negative children born to HIV-infected women who received anti-retroviral therapy during pregnancy (IARC, 2000). Our study, which for the first time verified the existence of human subjects hypersensitive to the genotoxic activity of AZT and the heritability of this hypersensitivity could be considered to minimize long-term mutagenic effects in treated subjects.
In conclusion, our study has demonstrated that the genotoxic effects of the telomerase inhibitor AZT on human peripheral lymphocytes was mainly regulated by genetic factors and identified hypersensitive subjects, possibly characterized by a high level of telomerase activity induced after PHA stimulation. Ageing did not modify the individual susceptibility to AZT induction of CA and MN, but nevertheless determined an overall increase in nuclear damage.
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Acknowledgements |
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We thank Prof. Giuseppe Novelli for the zygotism assessment and Prof. Maria Giulia Farace for helpful reading and discussion of the manuscript. We also thank Mr Graziano Bonelli for technical assistance. This work was supported by a MIUR grant to D.C., R.C. and P.P.
Declaration of interest
All of the authors of this work are currently conducting research sponsored by a grant from MURST (Ministero dell’Università e della Ricerca Scientifica e Tecnologica).
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2To whom correspondence should be addressed. Tel: +39 6 72596066; Fax: +39 6 72596053; Email: caporos{at}uniroma2.it
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