Another group of genes that might be involved in regulating the rate of aging comprises those whose mutations result in segmental progeroid syndromes. Such syndromes are characterized by the presence of a number of features similar to these of normal aging, but, in addition, each of them has its own specific signs and symptoms (Puzianowska-Kuznicka and Kuznicki 2005; Kudlow et al. 2007). On the molecular level, the majority of them are associated with impaired DNA repair, defects in transcription, and with genetic instability (de Boer et al. 2002; Andressoo and Hoeijmakers 2005; Puzianowska-Kuznicka and Kuznicki 2005; Neveling et al. 2007; Andressoo et al. 2009). Since normal senescent cells also have

The most extensively studied are three common XPD polymorphisms: the Arg156Arg C[A located in exon 6, the Asp312Asn G[A in exon 10, and the Lys751Gln A[C in exon 23. The majority of published data report correlations of these polymor- phisms with cancer risk (Wang et al. 2008). Although not completely consistent among themselves, these reports suggest that these polymorphisms might modify the risk of development of different cancer types including lung (Spitz et al. 2001; Kiyohara and Yoshimasu 2007; Hung et al. 2008), breast (Just- enhoven et al. 2004; Terry et al. 2004), skin (Vogel et al. 2001; Brewster et al. 2004; Millikan et al. 2006; Li et al. 2006), digestive tract (Ye et al. 2006; Wang

et al. 2008; Joshi et al. 2009) and blood (Wang et al. 2008) cancers.

from the Y group (22–37 years old, mean age 28.7 years old), in 18 people of middle age (M, 60– 69 years old, mean age 65.8 years old,), and in 17 extremely old subjects (90–100 years old, mean age 92.7 years old). All participants gave a written consent for participation in the study. The study protocol was approved by the Ethical Committee of the Medical University of Warsaw.

In this work we present evidence that certain Lys751Gln genetic variants of the XPD gene might be associated with the promotion of longevity, while the Arg156Arg and Asp312Asn polymorphisms show no such association in the Polish population. We also show that expression of the XPD gene, assessed in blood mononuclear cells, decreases in an age-depen- dent manner, which might be one of the reasons for the increase of genomic instability observed by other authors in senescent cells.

DNA isolation and restriction fragment length polymorphism analysis

Four milliliter blood samples were used for isolation of the genomic DNA that was performed using salting-out procedure (Miller et al. 1988). Genotyping of the selected polymorphism in the XPD was done by PCR amplification followed by digestion with an appropriate restriction enzyme (restriction fragment length polymorphism method—RFLP). PCR–RFLP conditions used for the analysis are shown in Table 1. The restriction fragments obtained were visualized on 2–3% agarose gels (Fig. 1a–c).

Study subjects

in two age groups: 149 Polish centenarians (C, 99.6– 107.2 years old, mean age 101.1 years old, 130 women, 19 men)—participants of the ‘‘PolStu99’’ study (Mossakowska et al. 2008). The control group consisted of 413 blood donors and volunteers (Y, 18– 45 years old, mean age 27.1 years old, 232 women, 181 men). Analysis of the XPD promoter methylation was performed in 10 subjects from the Y group (25– 37 years old, mean age 29.5 years old) and in 10 subjects from the C group (100–102.4 years old, mean age 101.4 years old), as well as in 10 additional subjects that formed another control group (M, 65.3– 66.5 years old, mean age 65.9 years old). Analysis of the XPD expression was performed in 19 subjects

Bisulfite treatment of DNA and DNA methylation analysis

Bisulfite modification of the genomic DNA was performed using EZ DNA Methylation-Gold Kit (Zymo Research Corporation, Orange, CA, USA). Five hundred nanogram of each genomic DNA was bisulfite-modified following the manufacturer’s pro- tocol with the only modification being the PCR incubation conditions; the samples were subjected to

Table 1 PCR–RFLP conditions used for the analysis of the Arg156Arg, Asp312Asn, and Lys751Gln polymorphisms in the XPD gene

Primers T MgCl PCR Restriction Alleles m 2 ((cid:3)C) (mM) product (bp) enzyme F: 50CAAACATCCTGTCCCTACTGG30 62 1.5 309 HinfI C: 217 bp, 92 bp R: 50CTCCTGCGATTAAAGGCTGT30 A: 221 bp F: 50GCCCCTGTCTGACTTGTCC30 56.7 1.5 204 Eco103I G: 204 bp R: 50GCTCACCCTGCAGCACTTCCT30a (StyI) A: 181 bp, 23 bp F: 50GAGGTTCAAGTTGTCCAAAACC30 61 1.5 346 PstI A: 237 bp, 109 bp R: 50ACAAGAGTGCCAGGGGTCAG30 C: 174 bp, 109 bp, 93 bp

a Underlined nucleotide is a mismatch introduced to create Eco103I (StyI) restriction T Melting temperature, F Forward, R Reverse, bp Base pairs m

of a Arg156Arg b Asp312Asn c Lys751Gln CC CA AA GA GG AA AC AA CC 309 237 204 217 181 174 109 93 92   

RNA isolation, reverse transcription, and real-time PCR

10 min and 53(cid:3)C for 30 min, then 8 cycles of 53(cid:3)C for 6 min, 37(cid:3)C for 30 min. Bisulfite-treated DNA was purified and resuspended in 10 ll of the M- elution buffer supplied by the manufacturer. Fifty nanogram (1 ll) of this DNA was used as a template in 12.5 ll PCR reaction performed with hot-start Platinum Taq polymerase (Invitrogen, Carlsbad, CA, USA) and with sense XPDsl 50GAGGATTAAATT TTAGTGAATGAGAT30 and antisense XPDasl 50AT AACCCCATCTTCAAAACCCCC30 primers. The PCR conditions were: initial denaturation at 94(cid:3)C for 3 min, 40 cycles of 94(cid:3)C for 30 s, 58(cid:3)C for 30 s, 72(cid:3)C for 1 min, final elongation at 72(cid:3)C for 10 min. The PCR product of 377 bp, corresponding to the XPD promoter fragment located on both sides of the transcription start site, was gel-purified and cloned into the pGEM-T vector (Promega, Madison, WI, USA). Three clones from each person were sequenced.