Sir2, a NAD-dependent deacetylase, modulates lifespan in yeasts, worms and flies. The SIRT1, mammalian homologue of Sir2, regulates signaling for favoring survival in stress. But whether SIRT1 has the function to influence cell viability and senescence under non-stressed conditions in human diploid fibroblasts is far from unknown. Our data showed that enforced SIRT1 expression promoted cell proliferation and antagonized cellular senescence with the characteristic features of delayed Senescence-Associated β-galactosidase (SA-β-gal) staining, reduced Senescence-Associated Heterochromatic Foci (SAHF) formation and G1 phase arrest, increased cell growth rate and extended cellular lifespan in human fibroblasts, while dominant-negative SIRT1 allele (H363Y) did not significantly affect cell growth and senescence but displayed a bit decreased lifespan.. Western blot results showed that SIRT1 reduced the expression of p16INK4A and promoted phosphorylation of Rb. Our data also exposed that overexpression of SIRT1 was accompanied by enhanced activation of ERK and S6K1 signaling. These effects were mimicked in both WI38 cells and 2BS cells by concentration-dependent resveratrol, a SIRT1 activator. It was noted that treatment of SIRT1-.transfected cells with Rapamycin, a mTOR inhibitor, reduced the phosphorylation of S6K1 and the expression of Id1, implying that SIRT1-induced phosphorylation of S6K1 may be partly for the decreased expression of p16INK4A and promoted phosphorylation of Rb in 2BS. It was also observed that the expression of SIRT1 and phosphorylation of ERK and S6K1 was declined in senescent 2BS. These findings suggested that SIRT1-promoted cell proliferation and antagonized cellular senescence in human diploid fibroblasts may be, in part, via the activation of ERK/ S6K1 signaling.

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We introduce two measures of connectivity that are applicable to standard GIS-based representations of street networks. The reach of a point measures the total street length covered by all paths extending out from that point that are no longer than a given threshold value. The directional distance of a street network from a point is measured according to the minimum number of direction changes required to reach any part of the network from that point, consistent with typical measures used in space syntax. However, our measure of directional distance requires no prior commitment as to the relational elements that make up the network. Any part of the network which is accessible from a point without a change of direction greater than a given threshold angle is treated as a single directional element for the purposes of computation. Street segments are characterized by the reach and directional distance of their midpoints. Networks are characterized by the average directional distance of the corresponding street segments. The measures render explicit the interplay between metric and topological properties of networks. Preliminary studies show that the measures discriminate well between different morphologies of street networks. When used to compare urban morphologies they are well correlated with standard measures used in the literature, with the added advantage that they can discriminate between street segments within the same urban area. Using field observations we also show that the measures can be used to model the effect of spatial configuration upon movement in ways which compare favorably to standard space syntax.

Id1, a member of Id family of helix-loop-helix transcriptional regulatory proteins, is implicated in cellular senescence by repressing p16INK4a expression, but the mechanisms and cellular effects in human diploid fibroblasts remain unknown. Here we analyzed the patterns of p16INK4a and Id1 expression during the lifespan of 2BS cells and presented the inverse correlation between these two proteins. Immunoprecipitation assays demonstrated the presence of endogenous interaction of Id1 and E47 proteins that was strong in young 2BS cells and weakened during replicative senescence and, thereby, influenced the transcription activation of p16INK4a by E47. Furthermore, we found that E47 protein could bind to the E-box-containing region in p16 INK4a promoter in senescent cells by chromatin immunoprecipitation analyses, suggesting that E47 is indeed ultimately involved in the regulation of p16INK4a transcription in vivo. Silencing Id1 expression in young cells by RNA interference induced an increased p16INK4a level and premature cellular senescence, whereas silencing E47 expression inhibited the expression of p16INK4a and delayed the onset of senescent phenotype. The present study demonstrated not only the capacity of Id1 to regulate p16INK4a gene expression by E47, but also the phenotypic consequence of the regulation on cellular senescence, moreover, raised the possibility of Id1-specific gene silencing for human cancer therapy.

Using a suppressive subtractive hybridization system, we identified CSIG (cellular senescence-inhibited gene protein; RSL1D1) that was abundant in young human diploid fibroblast cells but declined upon replicative senescence. Overexpression or knockdown of CSIG did not influence p21Cip1 and p16 INK4a expressions. Instead, CSIG negatively regulated PTEN and p27Kip1 expressions, in turn promoting cell proliferation. In PTEN-silenced HEK 293 cells and PTEN-deficient human glioblastoma U87MG cells, the effect of CSIG on p27 Kip1 expression and cell division was abolished, suggesting that PTEN was required for the role of CSIG on p27Kip1 regulation and cell cycle progression. Investigation into the underlying mechanism revealed that the regulation of PTEN by CSIG was achieved through a translational suppression mechanism. Further study showed that CSIG interacted with PTEN mRNA in the 5 untranslated region (UTR) and that knockdown of CSIG led to increased luciferase activity of a PTEN 5 UTR-luciferase reporter. Moreover, overexpression of CSIG significantly delayed the progression of replicative senescence, while knockdown of CSIG expression accelerated replicative senescence. Knockdown of PTEN diminished the effect of CSIG on cellular senescence. Our findings indicate that CSIG acts as a novel regulatory component of replicative senescence, which requires PTEN as a mediator and involves in a translational regulatory mechanism.

p16INK4a , a tumor suppressor gene that inhibits cyclindependent kinase 4 and cyclin-dependent kinase 6, is also implicated in the mechanisms underlying replicative senescence, because its RNA and protein accumulate as cells approach their finite number of population doublings in tissue culture. To further explore the involvement of p16INK4a in replicative senescence, we constructed a retroviral vector containing antisense p16INK4a , pDOR-ASp16, and introduced it into early passages of human diploid fibroblasts. The introduction of this construct significantly suppressed the expression of wild-type p16 INK4a . It also imposed a finite increase in proliferative life span and significant delay of several other cell senescent features, such as cell flattening, cell cycle arrest, and senescence-associated ␤-galactosidase positivity. Moreover, telomere shortening and decline in DNA repair capacity, which normally accompany cell senescence, are also postponed by the ASp16 transfection. The life span of fibroblasts was significantly extended, but the onset of replicative senescence could not be totally prevented. Telomerase could not be activated even though telomere shortening was slowed. These observations suggest that the telomere pathway of senescence cannot be bypassed by ASp16 expression. These data not only strongly support a role for p16INK4a in replicative senescence but also raise the possibility of using the antisense p16INK4a therapeutically.

Background p16 INK4a tumor suppressor protein has been widely proposed to mediate entrance of the cells into the senescent stage. Promoter of p16 INK4a gene contains at least five putative GC boxes, named GC-I to V, respectively. Our previous data showed that a potential Sp1 binding site, within the promoter region from −466 to −451, acts as a positive transcription regulatory element. These results led us to examine how Sp1 and/or Sp3 act on these GC boxes during aging in cultured human diploid fibroblasts.Methodology/Principal FindingsMutagenesis studies revealed that GC-I, II and IV, especially GC-II, are essential for p16 INK4a gene expression in senescent cells. Electrophoretic mobility shift assays (EMSA) and ChIP assays demonstrated that both Sp1 and Sp3 bind to these elements and the binding activity is enhanced in senescent cells. Ectopic overexpression of Sp1, but not Sp3, induced the transcription of p16 INK4a. Both Sp1 RNAi and Mithramycin, a DNA intercalating agent that interferes with Sp1 and Sp3 binding activities, reduced p16 INK4a gene expression. In addition, the enhanced binding of Sp1 to p16 INK4a promoter during cellular senescence appeared to be the result of increased Sp1 binding affinity, not an alteration in Sp1 protein level.Conclusions/SignificanceAll these results suggest that GC- II is the key site for Sp1 binding and increase of Sp1 binding activity rather than protein levels contributes to the induction of p16 INK4a expression during cell aging.

Peroxisome proliferator-activated receptor γ (PPARγ) plays an important role in the inhibition of cell growth by promoting cell-cycle arrest, and PPARγ activation induces the expression of p16INK4α (CDKN2A), an important cell-cycle inhibitor that can induce senescence. However, the role of PPARγ in cellular senescence is unknown. Here, we show that PPARγ promotes cellular senescence by inducing p16INK4α expression. We found several indications that PPARγ accelerates cellular senescence, including enhanced senescence-associated (SA)-β-galactosidase staining, increased G1 arrest and delayed cell growth in human fibroblasts. Western blotting studies demonstrated that PPARγ activation can upregulate the expression of p16INK4α. PPARγ can bind to the p16 promoter and induce its transcription, and, after treatment with a selective PPARγ agonist, we observed more-robust expression of p16INK4α in senescent cells than in young cells. In addition, our data indicate that phosphorylation of PPARγ decreased with increased cell passage. Our results provide a possible molecular mechanism underlying the regulation of cellular senescence.