Sir2 is a NAD+-dependent protein deacetylase that extends lifespan in yeast and worms. This study examines seven human proteins homologous to Sir2 (SIRT1 through SIRT7) for cellular localization, expression profiles, protein deacetylation activity, and effects on human cell lifespan. We found that: 1) three nuclear SIRT proteins (SIRT1, SIRT6, and SIRT7) show different subnuclear localizations: SIRT6 and SIRT7 are associated with heterochromatic regions and nucleoli, respectively, where yeast Sir2 functions; 2) SIRT3, SIRT4, and SIRT5 are localized in mitochondria, an organelle that links aging and energy metabolism; 3) cellular p53 is a major in vivo substrate of SIRT1 deacetylase, but not the other six SIRT proteins; 4) SIRT1, but not the other two nuclear SIRT proteins, shows an in vitro deacetylase activity on histone H4 and p53 peptides; and 5) overexpression of any one of the seven SIRT proteins does not extend cellular replicative lifespan in normal human fibroblasts or prostate epithelial cells. This study supports the notion that multiple human SIRT proteins have evolutionarily conserved and nonconserved functions at different cellular locations and reveals that the lifespan of normal human cells, in contrast to that of lower eukaryotes, cannot be manipulated by increased expression of a single SIRT protein.

Humans and animals undergo ageing, and although their primary cells undergo cellular senescence in culture, the relationship between these two processes is unclear. Here we show that gamma-H2AX foci (gamma-foci), which reveal DNA double-strand breaks (DSBs), accumulate in senescing human cell cultures and in ageing mice. They colocalize with DSB repair factors, but not significantly with telomeres. These cryptogenic gamma-foci remain after repair of radiation-induced gamma-foci, suggesting that they may represent DNA lesions with unrepairable DSBs. Thus, we conclude that accumulation of unrepairable DSBs may have a causal role in mammalian ageing.

Hypoxia induces angiogenesis and glycolysis for cell growth and survival, and also leads to growth arrest and apoptosis. HIF‐1α, a basic helix–loop–helix PAS transcription factor, acts as a master regulator of oxygen homeostasis by upregulating various genes under low oxygen tension. Although genetic studies have indicated the requirement of HIF‐1α for hypoxia‐induced growth arrest and activation of p21cip1, a key cyclin‐dependent kinase inhibitor controlling cell cycle checkpoint, the mechanism underlying p21cip1 activation has been elusive. Here we demonstrate that HIF‐1α, even in the absence of hypoxic signal, induces cell cycle arrest by functionally counteracting Myc, thereby derepressing p21cip1. The HIF‐1α antagonism is mediated by displacing Myc binding from p21cip1 promoter. Neither HIF‐1α transcriptional activity nor its DNA binding is essential for cell cycle arrest, indicating a divergent role for HIF‐1α. In keeping with its antagonism of Myc, HIF‐1α also downregulates Myc‐activated genes such as hTERT and BRCA1. Hence, we propose that Myc is an integral part of a novel HIF‐1α pathway, which regulates a distinct group of Myc target genes in response to hypoxia.

The finite proliferative potential of normal human cells leads to replicative cellular senescence, which is a critical barrier to tumour progression in vivo1–3. We show that human p53 isoforms (Δ133p53 and p53β)4 constitute an endogenous regulatory mechanism for p53-mediated replicative senescence. Induced p53β and diminished Δ133p53 were associated with replicative senescence, but not oncogene-induced senescence, in normal human fibroblasts. The replicatively senescent fibroblasts also expressed increased levels of miR-34a, a p53-induced microRNA5–9, the antisense inhibition of which delayed the onset of replicative senescence. The siRNA-mediated knockdown of endogenous Δ133p53 induced cellular senescence, which was attributed to the regulation of p21WAF1 and other p53 transcriptional target genes. In overexpression experiments, while p53β cooperated with full-length p53 to accelerate cellular senescence, Δ133p53 repressed miR-34a expression and extended cellular replicative lifespan, providing a functional connection of this microRNA to the p53 isoform-mediated regulation of senescence. The senescence-associated signature of p53 isoform expression (i.e., elevated p53β and reduced Δ133p53) was observed in vivo in colon adenomas with senescent phenotypes10, 11. The increased Δ133p53 and decreased p53β isoform expression found in colon carcinoma may signal an escape from the senescence barrier during the progression from adenoma to carcinoma.

Fifteen percent of lung cancer cases occur in never-smokers and show characteristics that are molecularly and clinically distinct from those in smokers. Epidermal growth factor receptor (EGFR) gene mutations, which are correlated with sensitivity to EGFR-tyrosine kinase inhibitors (EGFR-TKIs), are more frequent in never-smoker lung cancers. In this study, microRNA (miRNA) expression profiling of 28 cases of never-smoker lung cancer identified aberrantly expressed miRNAs, which were much fewer than in lung cancers of smokers and included miRNAs previously identified (e.g., up-regulated miR-21) and unidentified (e.g., down-regulated miR-138) in those smoker cases. The changes in expression of some of these miRNAs, including miR-21, were more remarkable in cases with EGFR mutations than in those without these mutations. A significant correlation between phosphorylated-EGFR (p-EGFR) and miR-21 levels in lung carcinoma cell lines and the suppression of miR-21 by an EGFR-TKI, AG1478, suggest that the EGFR signaling is a pathway positively regulating miR-21 expression. In the never-smoker-derived lung adenocarcinoma cell line H3255 with mutant EGFR and high levels of p-EGFR and miR-21, antisense inhibition of miR-21 enhanced AG1478-induced apoptosis. In a never-smoker-derived adenocarcinoma cell line H441 with wildtype EGFR, the antisense miR-21 not only showed the additive effect with AG1478 but also induced apoptosis by itself. These results suggest that aberrantly increased expression of miR-21, which is enhanced further by the activated EGFR signaling pathway, plays a significant role in lung carcinogenesis in never-smokers, as well as in smokers, and is a potential therapeutic target in both EGFR-mutant and wild-type cases.apoptosis ͉ microRNA ͉ microarray ͉ EGFR-TKI ͉ therapeutic target

The E6 and E7 oncogenes of human papillomavirus type 16 (HPV-16) are sufficient for the immortalization of human genital keratinocytes in vitro. The products of these viral genes associate with p53 and pRb tumor suppressor proteins, respectively, and interfere with their normal growth-regulatory functions. The HPV-16 E6 protein has also been shown to increase the telomerase enzyme activity in primary epithelial cells by an unknown mechanism. We report here that a study using reverse transcription-PCR and RNase protection assays in transduced primary human foreskin keratinocytes (HFKs) shows that the E6 gene (but not the E7 gene) increases telomerase hTERT gene transcription coordinately with E6-induced telomerase activity. In these same cells, the E6 gene induces a 6.5-fold increase in the activity of a 1,165-bp 5 promoter/regulatory region of the hTERT gene, and this induction is attributable to a minimal 251-bp sequence (؊211 to ؉40). Furthermore, there is a 35-bp region (؉5 to ؉40) within this minimal E6-responsive promoter that is responsible for 60% of E6 activity. Although the minimal hTERT promoter contains Myc-responsive E-box elements and recent studies have suggested a role for Myc protein in hTERT transcriptional control, we found no alterations in the abundance of either c-Myc or c-Mad in E6-transduced HFKs, suggesting that there are other or additional transcription factors critical for regulating hTERT expression.The human papillomaviruses (HPVs) designated as "high risk" types, such as HPV type 16 (HPV-16) and HPV-18, are associated with anogenital tract lesions that can progress to malignancy (44, 45). The E6 and E7 viral genes appear to be responsible for both the in vivo and in vitro transforming activity of these high-risk viruses (24, 46), and each of these genes can independently transform established rodent cell lines (3,29,39). Interestingly, the E6 gene can independently immortalize primary human mammary epithelial cells in culture (2).The transforming activities of the E6 and E7 viral gene products reside in their ability to interact specifically with cellular regulatory proteins and interfere with their normal functioning. The E7 protein interacts with pRb and abrogates its tumor-suppressive activity (8,25), while the E6 protein cooperates with E6AP, a ubiquitin E3 ligase, to target p53 tumor suppressor protein for ubiquitin-dependent degradation (16,31,32,42). Other less well characterized functions for E6 oncoprotein have been proposed (9,18,22), including the activation of telomerase (20), which is a ribonucleoprotein enzyme important for the maintenance of telomeric structures at the ends of chromosomes (10, 27).Telomerase activity is detected in more than 90% of immortalized and cancer cells but absent in most normal somatic cells (17, 23), suggesting that telomerase activation is an important event during the process of immortalization and malignant transformation. The absence of telomerase activity in normal cells results in progressive telomere erosion with each cell cycle due t...

Nutlin-3, an MDM2 inhibitor, activates p53, resulting in several types of cancer cells undergoing apoptosis. Although p53 is mutated or deleted in f50% of all cancers, p53 is still functionally active in the other 50%. Consequently, nutlin-3 and similar drugs could be candidates for neoadjuvant therapy in cancers with a functional p53. Cellular senescence is also a phenotype induced by p53 activation and plays a critical role in protecting against tumor development. In this report, we found that nutlin-3a can induce senescence in normal human fibroblasts. Nutlin-3a activated and repressed a large number of p53-dependent genes, including those encoding microRNAs. mir-34a, mir-34b, and mir-34c, which have recently been shown to be downstream effectors of p53-mediated senescence, were up-regulated, and inhibitor of growth 2 (ING2) expression was suppressed by nutlin-3a treatment. Two candidates for a p53-DNA binding consensus sequence were found in the ING2 promoter regulatory region; thus, we performed chromatin immunoprecipitation and electrophoretic mobility shift assays and confirmed p53 binding directly to those sites. In addition, the luciferase activity of a construct containing the ING2 regulatory region was repressed after p53 activation. Antisense knockdown of ING2 induces p53-independent senescence, whereas overexpression of ING2 induces p53-dependent senescence. Taken together, we conclude that nutlin-3a induces senescence through p53 activation in normal human fibroblasts, and p53-mediated mir34a, mir34b, and mir34c upregulation and ING2 down-regulation may be involved in the senescence pathway. [Cancer Res 2008;68(9):3193-203]

SummaryAccumulation of DNA damage may play an essential role in both cellular senescence and organismal aging. The ability of cells to sense and repair DNA damage declines with age. However, the underlying molecular mechanism for this age-dependent decline is still elusive. To understand quantitative and qualitative changes in the DNA damage response during human aging, DNA damageinduced foci of phosphorylated histone H2AX (γ γ γ γ -H2AX), which occurs specifically at sites of DNA double-strand breaks (DSBs) and eroded telomeres, were examined in human young and senescing fibroblasts, and in lymphocytes of peripheral blood. Here, we show that the incidence of endogenous γ γ γ γ -H2AX foci increases with age. Fibroblasts taken from patients with Werner syndrome, a disorder associated with premature aging, genomic instability and increased incidence of cancer, exhibited considerably higher incidence of γ γ γ γ -H2AX foci than those taken from normal donors of comparable age. Further increases in γ γ γ γ -H2AX focal incidence occurred in culture as both normal and Werner syndrome fibroblasts progressed toward senescence. The rates of recruitment of DSB repair proteins to γ γ γ γ -H2AX foci correlated inversely with age for both normal and Werner syndrome donors, perhaps due in part to the slower growth of γ γ γ γ -H2AX foci in older donors. Because genomic stability may depend on the efficient processing of DSBs, and hence the rapid formation of γ γ γ γ -H2AX foci and the rapid accumulation of DSB repair proteins on these foci at sites of nascent DSBs, our findings suggest that decreasing efficiency in these processes may contribute to genome instability associated with normal and pathological aging.