Cellular senescence can modulate various pathologies and is associated with irreparable DNA double-strand breaks (IrrDSBs). Extracellular senescence metabolomes (ESMs) were generated from fibroblasts rendered senescent by proliferative exhaustion (PEsen) or 20 Gy of γ rays (IrrDSBsen) and compared with those of young proliferating cells, confluent cells, quiescent cells, and cells exposed to repairable levels of DNA damage to identify novel noninvasive markers of senescent cells. ESMs of PEsen and IrrDSBsen overlapped and showed increased levels of citrate, molecules involved in oxidative stress, a sterol, monohydroxylipids, tryptophan metabolism, phospholipid, and nucleotide catabolism, as well as reduced levels of dipeptides containing branched chain amino acids. The ESM overlaps with the aging and disease body fluid metabolomes, supporting their utility in the noninvasive detection of human senescent cells in vivo and by implication the detection of a variety of human pathologies. Intracellular metabolites of senescent cells showed a relative increase in glycolysis, gluconeogenesis, the pentose-phosphate pathway, and, consistent with this, pyruvate dehydrogenase kinase transcripts. In contrast, tricarboxylic acid cycle enzyme transcript levels were unchanged and their metabolites were depleted. These results are surprising because glycolysis antagonizes senescence entry but are consistent with established senescent cells entering a state of low oxidative stress.

Fibrosis can occur in many organs, where it is a debilitating and preneoplastic condition. The senescence of activated fibroblasts has been proposed to ameliorate fibrosis via the innate immune system but its role in humans has not been investigated. The availability of oral submucous fibrosis (OSMF) biopsies at different stages of disease progression allowed us to test the hypothesis that senescent fibroblasts accumulate with the progression of human fibrosis in vivo, and also to examine the mechanism of senescence. We tested the hypothesis that senescent cells may ameliorate fibrosis by increasing the secretion of matrix metalloproteinases (MMPs). We have used a combination of in situ immunodetection techniques, drug treatments, fluorescence-activated cell sorting and enzyme-linked absorbance assays on tissue samples and fibroblast cultures. We report a novel panning technique, based on fibronectin adhesion rates, to enrich and deplete senescent cells from fibroblast populations. Senescent fibroblasts, as determined by the presence of senescence-associated heterochromatic foci, accumulated with OSMF progression (R(2) = 0.98) and possessed a reduced replicative lifespan in vitro. Unlike wounds, however, OSMF fibroblasts were quiescent in vivo and consistent with this observation, possessed functional telomeres of normal length. Senescence was associated in vivo and in vitro with oxidative damage, DNA damage foci and p16(INK4A) accumulation and required the production of reactive oxygen species (ROS), perhaps from damaged mitochondria, but not the continuous presence of the disease stimulus (areca nut and tobacco), the tissue environment or other cell types. Depletion of OSMF fibroblasts of senescent cells showed that these cells accounted for 25-83 times more MMP-1 and -2 than their pre-senescent counterparts. The results show that the accumulation of senescent fibroblasts in human fibrosis occurs by a telomere-independent mechanism involving ROS and may locally ameliorate the condition by the increased expression of MMPs prior to clearance by the immune system.

Telomere length is maintained by the enzyme, telomerase, which has been linked to cellular immortality and tumour progression. However, the reasons for the high levels of telomerase found in human tumours are unknown. We have mapped the human telomerase RNA gene, (hTR), to chromosome 3q26.3 and show the hTR gene to be ampli®ed in four carcinomas, (2/33 cervix, 1/31 head and neck, 1/9 lung). In addition, increased copy numbers of the hTR locus was also observed in 97% of tumours. By in situ hybridisation, the histological distribution of high levels of hTR expression could be demonstrated in a lung tumour and its metastasis with hTR ampli®cation. These results are the ®rst report of genetic alterations involving a known component of telomerase in human cancer. Indeed, it is also the ®rst report of the ampli®cation of a speci®c locus within the chromosome 3q region frequently subject to copy number gains in human tumours. In addition, we also show for the ®rst time the histological distribution of the RNA component of telomerase in human tumours.

Background:Previous studies have demonstrated that senescent cancer-associated fibroblasts (CAFs) derived from genetically unstable oral squamous cell carcinomas (GU-OSCC), unlike non-senescent CAFs from genetically stable carcinomas (GS-OSCC), promoted keratinocyte invasion in vitro in a paracrine manner. The mechanism by which this occurs is unclear.Methods:Previous work to characterise the senescent-associated secretory phenotype (SASP) has used antibody arrays, technology that is limited by the availability of suitable antibodies. To extend this work in an unbiased manner, we used 2D gel electrophoresis and mass spectroscopy for protein identification. Matrix metalloproteinases (MMPs) were investigated by gelatin zymography and western blotting. Neutralising antibodies were used to block key molecules in the functional assays of keratinocyte adhesion and invasion.Results:Among a variety of proteins that were differentially expressed between CAFs from GU-OSCC and GS-OSCC, MMP-2 was a major constituent of senescent CAF-CM derived from GU-OSCC. The presence of active MMP-2 was confirmed by gelatine zymography. MMP-2 derived from senescent CAF-CM induced keratinocyte dis-cohesion and epithelial invasion into collagen gels in a TGF-β-dependent manner.Conclusions:Senescent CAFs from GU-OSCC promote a more aggressive oral cancer phenotype by production of active MMP-2, disruption of epithelial adhesion and induction of keratinocyte invasion.

Ectopic expression of telomerase blocks both telomeric attrition and senescence, suggesting that telomeric attrition is a mitotic counting mechanism that culminates in replicative senescence. By holding human ®broblast cultures con¯uent for up to 12 weeks at a time, we con®rmed previous observations and showed that telomeric attrition requires cell division and also, that senescence occurs at a constant average telomere length, not at a constant time point. However, on resuming cell division, these long-term con¯uent (LTC) cultures completed 15 ± 25 fewer mean population doublings (MPDs) than the controls prior to senescence. These lost divisions were mainly accounted for by slow cell turnover of the LTC cultures and by permanent cell cycle exit of 94% of the LTC cells, which resulted in many cell divisions being unmeasured by the MPD method. In the LTC cultures, p27 KIP1 accumulated and pRb became under-phosphorylated and under-expressed. Also, coincident with permanent cell cycle exit and before 1 MPD was completed, the LTC cultures upregulated the cell cycle inhibitors p21 WAF and p16 INK4A but not p14 ARF and developed other markers of senescence. We then tested the relationship between cell cycle re-entry and the cell cycle-inhibitory proteins following subculture of the LTC cultures. In these cultures, the downregulation of p27 KIP1 and the phosphorylation of pRb preceded the complete resumption of normal proliferation rate, which was accompanied by the down-regulation of p16 INK4A . Our results show that most normal human ®broblasts can accumulate p16 INK4A , p21 WAF and p27 KIP1 and senesce by cell division-independent mechanism(s). Furthermore, this form of senescence likely requires p16 INK4A and perhaps p27 KIP1 . Oncogene (2001) 20, 3541 ± 3552.