Do senescence markers correlate in vitro and in situ within individual human donors?

Molecular Epidemiolog

Systemic age-associated DNA hypermethylation of ELOVL2 gene : in vivo and in vitro evidences of a cell replication process

Epigenetic remodeling is one of the major features of the aging process. We recently demonstrated that DNA methylation of ELOVL2 and FHL2 CpG islands is highly correlated with age in whole blood. Here we investigated several aspects of age-associated hypermethylation of ELOVL2 and FHL2 We showed that ELOVL2 methylation is significantly different in primary dermal fibroblast cultures from donors of different ages. Using epigenomic data from public resources, we demonstrated that most of the tissues show ELOVL2 and FHL2 hypermethylation with age. Interestingly, ELOVL2 hypermethylation was not found in tissues with very low replication rate. We demonstrated that ELOVL2 hypermethylation is associated with in vitro cell replication rather than with senescence. We confirmed intra-individual hypermethylation of ELOVL2 and FHL2 in longitudinally assessed participants from the Doetinchem Cohort Study. Finally we showed that, although the methylation of the two loci is not associated with longevity/mortality in the Leiden Longevity Study, ELOVL2 methylation is associated with cytomegalovirus status in nonagenarians, which could be informative of a higher number of replication events in a fraction of whole-blood cells. Collectively, these results indicate that ELOVL2 methylation is a marker of cell divisions occurring during human aging

Kinetic profiling of the c-Myc transcriptome and bioinformatic analysis of repressed gene promoters

10.4161/cc.10.13.16249Cell Cycle10132184-219

DNA damage markers in dermal fibroblasts in vitro reflect chronological donor age

Molecular Epidemiolog

Characterization of IkappaB kinases - IkappaB-a is not phosphorylated by Raf-1 or protein kinase C isozymes, but is a casein kinase II substrate.

The NF-kappaB transcription factor is activated by a wide variety of stimuli, including phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate. In its inactive state, NF-kappaB is sequestered in the cytoplasm tethered to an inhibitor protein, IkappaB. Activation comprises the rapid phosphorylation of IkappaB-alpha at N-terminal sites, which presumably marks IkappaB-alpha for proteolytic degradation and leads to release of NF-kappaB into the nucleus. In addition, IkappaB-alpha is constitutively phosphorylated at the C terminus, which may be a prerequisite for proper IkappaB function. Protein kinase C (PKC) is activated by 12-O-tetradecanoylphorbol-13-acetate and has been previously reported to phosphorylate IkappaB-alpha in vitro. As PKC has turned out to constitute a multigene family encoding isozymes with different biological functions, we have reinvestigated IkappaB-alpha phosphorylation by PKC using recombinant PKC isozymes expressed in insect cells. While crude PKC preparations were efficient IkappaB-alpha kinases, highly purified PKC isozymes completely failed to phosphorylate IkappaB-alpha. Biochemical separation of porcine spleen yielded at least two fractions with IkappaB-alpha kinase activity, both of which were devoid of detectable PKC isozymes. One peak contained both Raf-1 and casein kinase II (CKII). Purified Raf-1 does not phosphorylate IkappaB-alpha directly, but associates with CKII, which efficiently phosphorylates the C terminus of IkappaB-alpha. Two-dimensional phosphopeptide mapping and high pressure liquid chromatography-mass spectroscopy analysis showed that all IkappaB-alpha kinases induced phosphorylation at the same prominent sites in the C terminus. Our results clearly indicate that PKC isozymes alpha, beta, gamma, delta, epsilon, eta, and zeta as well as Raf-1 are not IkappaB-alpha kinases. They furthermore demonstrate that IkappaB-alpha is targeted by several kinases, one of which appears to be CKII

The RKIP (Raf-1 Kinase Inhibitor Protein) conserved pocket binds to the phosphorylated N-region of Raf-1 and inhibits the Raf-1-mediated activated phosphorylation of MEK

The Raf-MEK-ERK pathway regulates many fundamental biological processes, and its activity is finely tuned at multiple levels. The Raf kinase inhibitory protein (RKIP) is a widely expressed negative modulator of the Raf-MEK-ERK signaling pathway. We have previously shown that RKIP inhibits the phosphorylation of MEK by Raf-1 through interfering with the formation of a kinase-substrate complex by direct binding to both Raf-1 and MEK. Here, we show that the evolutionarily conserved ligand-binding pocket of RKIP is required for its inhibitory activity towards the Raf-1 kinase mediated activation of MEK. Single amino acid substitutions of two of the conserved residues form the base and the wall of the pocket confers a loss-of-function phenotype on RKIP. Loss-of-function RKIP mutants still appear to bind to Raf-1. However the stability of the complexes formed between mutants and the N-region Raf-1 phosphopeptide were drastically reduced. Our results therefore suggest that the RKIP conserved pocket may constitute a novel phosphoamino-acid binding motif and is absolutely required for RKIP function. (C) 2008 Elsevier Inc. All rights reserve

The number of p16INK4a positive cells in human skin reflects biological age

Pathophysiology, epidemiology and therapy of agein