Feedback between p21 and reactive oxygen production is necessary for cell senescence.

Cellular senescence--the permanent arrest of cycling in normally proliferating cells such as fibroblasts--contributes both to age-related loss of mammalian tissue homeostasis and acts as a tumour suppressor mechanism. The pathways leading to establishment of senescence are proving to be more complex than was previously envisaged. Combining in-silico interactome analysis and functional target gene inhibition, stochastic modelling and live cell microscopy, we show here that there exists a dynamic feedback loop that is triggered by a DNA damage response (DDR) and, which after a delay of several days, locks the cell into an actively maintained state of 'deep' cellular senescence. The essential feature of the loop is that long-term activation of the checkpoint gene CDKN1A (p21) induces mitochondrial dysfunction and production of reactive oxygen species (ROS) through serial signalling through GADD45-MAPK14(p38MAPK)-GRB2-TGFBR2-TGFbeta. These ROS in turn replenish short-lived DNA damage foci and maintain an ongoing DDR. We show that this loop is both necessary and sufficient for the stability of growth arrest during the establishment of the senescent phenotype.

Vulnerability to oxidative stress and different patterns of senescence in human peritoneal mesothelial cell strains.

Both the ascites fluid-derived mesothelial cell line LP-9 and primary cultures of human omentum-derived mesothelial cells (HOMCs) are commonly used in experimental studies. However, they seem to have a different replicative potential in vitro. In the present study, we have attempted to determine the causes of this discrepancy. HOMCs were found to divide fewer times and enter senescence earlier than LP-9 cells. This effect was coupled with earlier increases in the expression of senescence-associated-beta-galactosidase and cell cycle inhibitors p16INK4a and p21WAF1. Moreover, almost 3 times as many early-passage HOMCs as LP-9 cells bore senescence-associated DNA damage foci. In sharp contrast to LP-9 cells, the foci present in HOMCs localized predominantly outside the telomeres, and the HOMC telomere length did not significantly shorten during senescence. Compared with LP-9 cells, HOMCs were found to enter senescence with significantly lower levels of lipofuscin and damaged DNA, and markedly decreased glutathione contents. In addition, early-passage HOMCs generated significantly more reactive oxygen species either spontaneously or in response to exogenous oxidants. These results indicate that compared with LP-9 cells, HOMCs undergo stress-induced telomere-independent premature senescence, which may result from increased vulnerability to oxidative DNA injury.

Mitochondrial dysfunction is a possible cause of accelerated senescence of mesothelial cells exposed to high glucose.

High glucose has been found to accelerate cell senescence in vitro. The exact mechanism of this effect is, however, still poorly understood. In this paper we show that human peritoneal mesothelial cells (HPMCs) propagated under high (30mM) glucose were characterized by higher density of DNA double-strand breaks than cells exposed to standard (5mM) glucose concentration. Under both low and high glucose conditions, the vast majority of DNA damage localized to non-telomeric regions of the genome. Moreover, exposure to high glucose resulted in increased accumulation of lipofuscin, increased production of superoxides and peroxides as well as reduced mitochondrial membrane potential and increased mitochondrial mass. Treatment of cells with the free radical scavenger PBN partially rescued the premature senescence caused by high glucose. Together, these results indicate that high glucose may accelerate senescence of HPMCs by impairing mitochondrial function, resulting in overproduction of reactive oxygen species and extensive DNA damage.

Premature senescence of mesothelial cells is associated with non-telomeric DNA damage.

Human peritoneal mesothelial cells (HPMCs) senesce in vitro after barely few population doublings. In this report, we show that senescence of HPMCs is associated with increased accumulation of gamma-H2A.X foci, which reveal DNA double-strand breaks. Of note, already early-passage cultures contain a considerable fraction (44+/-10%) of cells bearing gamma-H2A.X foci. The gamma-H2A.X foci localize predominantly to non-telomeric DNA, either in young or senescent cells. Moreover, HPMCs seem to have unusually short telomeres (approximately 3.5 kbp) despite the presence of active telomerase. These telomeres do not shorten during senescence, but the activity of telomerase decreases to undetectable levels. In addition, senescence of HPMCs is associated with mitochondrial dysfunction, as manifested by increased production of reactive oxygen species and reduced mitochondrial membrane potential. These results may indicate that premature senescence of HPMCs is largely related to oxidative stress-induced DNA damage in non-telomeric regions of the genome.

TRF2 overexpression diminishes repair of telomeric single-strand breaks and accelerates telomere shortening in human fibroblasts.

Repair of single strand breaks in telomeric DNA is less efficient than in other genomic regions. This leads to an increased vulnerability of telomeric DNA towards damage induced by reactive oxygen species (ROS) and to accelerated telomere shortening under oxidative stress. The causes for the diminished repair efficacy in telomeres are unknown. We show here that overexpression of the telomere-binding protein TRF2 further reduces telomeric, but not genomic, single strand break repair. This suggests the possibility of strand break repair in telomeres being sterically hindered by the three-dimensional structure of the telomere DNA-protein complex and explains the effect of TRF2 on telomere shortening rates in telomerase-negative cells.

Additive cytotoxic effect of apoptin and chemotherapeutic agents paclitaxel and etoposide on human tumour cells.

Gene therapy experiments in animal models have shown that apoptin expression results in tumour regression without any significant side effects. Therefore, apoptin is regarded as a potential anticancer drug for clinical applications. In this study, we analysed whether chemotherapeutic agents combined with apoptin treatment could result in enhanced cytotoxicity in human tumour cell cultures. Combined treatment with recombinant adenovirus AdAptVP3 expressing apoptin and etoposide clearly showed an additive cytotoxic effect on human osteosarcoma U2OS cells. Paclitaxel treatment combined with apoptin expression significantly inhibited the survival of p53-positive human osteosarcoma U2OS and non-small lung carcinoma A549 cells, p53-negative human osteosarcoma Saos-2 cells and p53-mutant human prostate cancer Du145 cells, already at low doses of the chemotherapeutic agent. Our results indicate that the cytotoxicity-enhancing action by the tumour-specific apoptin in combination with chemotherapeutic agents might offer an effective and safe antitumour therapeutics.

Epigenetic inactivation of the human sprouty2 (hSPRY2) homologue in prostate cancer.

Abnormal signalling events mediated by receptor tyrosine kinases (RTKs) contribute to human carcinogenesis. Sprouty2 (Spry2) is a key antagonistic regulator of RTK signalling and suppression of its expression or function may facilitate proliferation and angiogenesis. Using prostate cancer (CaP) as a model, we investigated the significance of Spry2 in human malignancy. We observed downregulated Spry2 expression in invasive CaP cell lines and high-grade clinical CaP (compared to benign prostatic hyperplasia (BPH) and well-differentiated tumours, P=0.041). A large CpG island is associated with hSPRY2, and extensive hypermethylation of this CpG island was observed in 76-82% of high-grade CaP, while control BPH tissues were predominantly unmethylated (P=0.0005). Furthermore, suppressed Spry2 expression correlated with methylation of the CpG region in clinical samples (P=0.004) and treatment with 5-aza-2'-deoxycytidine reactivated Spry2 expression in LNCaP and PC-3M cells. hSPRY2 maps to the long arm of chromosome 13 (13q31.1), where loss of heterozygosity (LOH) has been reported. We found no evidence of mutation; however, we demonstrated 27-40% LOH using flanking markers to hSPRY2. Hence, while biallelic epigenetic inactivation of hSPRY2 represents the main genetic event in prostate carcinogenesis, the observed 27-40% LOH presents evidence of hemizygous deletion with the remaining allele hypermethylated. We therefore propose hSPRY2 as a potential tumour suppressor locus in CaP.