Increased poly(ADP-ribose) polymerase activity in lymphoblastoid cell lines from centenarians.

Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins which is catalyzed by poly(ADP-ribose) polymerase and represents an immediate response of eukaryotic cells to oxidative and other types of DNA damage. Previously a strong correlation had been detected between maximal poly(ADP-ribose) polymerase activity in permeabilized mononuclear leukocytes of various mammalian species and species-specific life span. To study a possible relation between longevity and poly(ADP-ribosyl)ation in humans we measured maximal oligonucleotide-stimulated poly(ADP-ribose) polymerase activity in permeabilized, Epstein-Barr virus transformed lymphoblastoid cell lines from a French population of 49 centenarians and 51 controls aged 20-70 years. Maximal enzyme activity was significantly higher in centenarians than in controls [median of controls: 9035 cpm/10(6) cells (lower quartile: 6156; upper quartile: 11,410); median of centenarians: 10,380 cpm/10(6) cells (lower quartile: 7994; upper quartile: 12,991); P=0.031 by Mann-Whitney U test]. In a subset of 16 controls and 24 centenarians, cellular poly(ADP-ribose) polymerase content was determined by quantitative western blotting, thus allowing the calculation of specific enzyme activity. The latter was significantly higher in centenarians (P=0.006), the median value for centenarians being about 1.6-fold that of controls. Specific poly(ADP-ribose) polymerase activity was a more powerful parameter for differentiating between centenarians and controls than enzyme activity relative to cell number. In addition, in a genetic association study we analyzed 437 DNA samples (239 centenarians and 198 controls) by PCR amplification of a polymorphic dinucleotide repeat located in the promoter region of the poly(ADP-ribose) polymerase gene in an attempt to detect an association between this polymorphic marker and variability of enzyme activity or human longevity. However, this genetic analysis revealed no significant enrichment of any of the alleles or genotypes identified among centenarians or controls, but its power was limited by the relatively weak heterozygosity of this polymorphic marker in our population (51%). Viewed together with previous results on poly(ADP-ribose) polymerase activity in various mammalian species, the present data provide further evidence for the notion that longevity is associated with a high poly(ADP-ribosyl)ation capacity.

New polymorphisms in the human poly(ADP-ribose) polymerase-1 coding sequence: lack of association with longevity or with increased cellular poly(ADP-ribosyl)ation capacity.

Poly(ADP-ribose) polymerase-1 (PARP-1) encoded by the PARP-1 gene, is a ubiquitous and abundant DNA-binding protein involved in the cellular response to various genotoxic agents. In a previous study we showed that maximal oligonucleotide-stimulated poly(ADP-ribosyl)ation was significantly higher in permeabilised lymphoblastoid cell lines from a French population of centenarians compared with controls aged 20-70 years, supporting the notion that longevity is associated with a genetically determined, high poly(ADP-ribosyl)ation capacity. Here, we describe four new genetic polymorphisms, three of which represent silent nucleotide variants (C402T, T1011C, G1215A), and one of which leads to a valine762-to-alanine exchange (T2444C). We undertook an association study between two of these polymorphisms and human longevity or poly(ADP-ribosyl)ation capacity in permeabilised lymphoblastoid cells. By analysing 648 DNA samples from a French population (324 centenarians and 324 controls) by fluorescent-allele-specific PCR, we showed the absence of any significant enrichment of any of the genotypes in the study of centenarians versus controls. Furthermore, we studied genotype distributions from individuals who had previously been tested for poly(ADP-ribosyl)ation capacity. None of the genotype combinations at any polymorphic site studied could be related to a high or low level of poly(ADP-ribosyl)ation capacity. Together, these results strongly suggest that the longevity-related differences in the poly(ADP-ribosyl)ation capacity of human lymphoblastoid cell lines cannot be explained by genetic polymorphisms in the PARP-1 coding sequence and that other mechanisms have to be considered as potential regulators of specific poly(ADP-ribosyl)ation capacity.

Modulation of DNA breakage induced via the Fenton reaction.

The conversion of the covalently closed circular double-stranded supercoiled DNA (pBR322) to a relaxed circle was used to investigate DNA nicking induced by Fe2+ and H2O2. In our experimental conditions of ionic strength (150 mM NaCl), pH = 7 and temperature (37 degrees C), the dose-response curve for the ferrous iron mediated H2O2 dependent DNA nicking is peculiar. For a fixed concentration of ferrous iron (2 microM), the concentration of H2O2 producing a maximum extent of DNA nicking was about 10-30 microM. The DNA single-strand breakage decreased with an increase of H2O2 concentration. We have investigated the effects of several factors such as the nature of the buffer, ionic strength, temperature and pH. Buffer components leading to the autoxidation of ferrous iron to ferric iron (phosphate) or to the scavenging of reactive oxygen species (Tris) greatly alter the dose-response curve. The H2O2 concentrations required for producing the maximum extent of DNA single-strand breaks at 4 degrees C and 56 degrees C were respectively 30 microM and 3 microM. At pH = 10, the pattern of the dose-response curve was totally different. The data showed that the peculiar dose-response curve for the ferrous iron mediated H2O2 dependent DNA nicking greatly depended on the experimental conditions.

Lack of association between human longevity and genetic polymorphisms in drug-metabolizing enzymes at the NAT2, GSTM1 and CYP2D6 loci.

In the present study, the possible role of genetic polymorphism of three drug-metabolizing enzymes, debrisoquine/sparteine hydroxylase (CYP2D6), glutathione S-transferase mu (GSTM1), and N-acetyltransferase (NAT2), as a putative genetic component of human longevity, was explored. A total of 817 DNA samples from a centenarian and a control (20-70 years) population was subjected to PCR-coupled RFLP methods. Subjects were genotyped for the CYP2D6*3 (A2637 deletion) and CYP2D6*4 (G1934A transition) alleles, for four mutations of NAT2 [namely, NAT2*5A (C481T), NAT2*6A (G590A), NAT2*7A (G857A), and NAT2*14A (G191A)], and for the presence or absence of GSTM1 gene deletion. No significant difference was found at these three loci between centenarian and control subjects with respect to allelic variant frequencies, genotype distributions or predicted phenotypes deduced from genotype combinations. By comparing the distribution of combined genotypes for the polymorphisms tested at the CYP2D6, NAT2, and GSTM1 loci, none of the predicted phenotypes concerning debrisoquine hydroxylase extensive-metabolizer or poor-metabolizer phenotypes, slow or fast N-acetylation capacities, and active or defective glutathione S-transferase, could be correlated with human longevity, alone or in combination.