From the Hayflick mosaic to the mosaics of ageing. Role of stress-induced premature senescence in human ageing

The Hayflick limit-senescence of proliferative cell types-is a fundamental feature of proliferative cells in vitro. Various human proliferative cell types exposed in vitro to many types of subcytotoxic stresses undergo stress-induced premature senescence (SIPS) (also called stress-induced premature senescence-like phenotype, according to the definition of senescence). The known mechanisms of appearance the main features of SIPS are reviewed: senescent-like morphology, growth arrest, senescence-related changes in gene expression, telomere shortening. Long before telomere-shortening induces senescence, other factors such as culture conditions or lack of 'feeder cells' can trigger either SIPS or prolonged reversible G(0) phase of the cell cycle. In vivo, 'proliferative' cell types of aged individuals are likely to compose a mosaic made of cells irreversibly growth arrested or not. The higher level of stress to which these cells have been exposed throughout their life span, the higher proportion of the cells of this mosaic will be in SIPS rather than in telomere-shortening dependent senescence. All cell types undergoing SIPS in vivo, most notably the ones in stressful conditions, are likely to participate in the tissular changes observed along ageing. For instance, human diploid fibroblasts (HDFs) exposed in vivo and in vitro to pro-inflammatory cytokines display biomarkers of senescence and might participate in the degradation of the extracellular matrix observed in ageing.     

Spontaneous and induced activation of rat oocytes

Ovulated rat oocytes undergo spontaneous activation during in vitro culture. After extrusion of the second polar body, they do not enter interphase but are arrested again in next metaphase-like stage (M III arrest). The present study demonstrates that puromycin and chloral hydrate can trigger transition to interphase of metaphase II and spontaneously (incompletely) activated rat oocytes. The response of oocytes to these activators depends on their stage at the time of application of a stimulus. Metaphase II oocytes enter interphase at 86.8% when treated with puromycin and in 28.7% after chloral hydrate activation. Oocytes activated with chloral hydrate at the time of spontaneously induced anaphase II enter interphase at 64.8%, but after reaching the stage of telophase II their capability to shift to interphase is again low (28.8%). Finally, M III oocytes cannot be forced to enter interphase by either chloral hydrate or puromycin treatment. This study shows that resumption of the second meiotic division and transition to interphase--the two processes that normally occur in succession as a response to oocyte activatin--can be experimentally separated.     

Cell cycle arrest associated with anoxia-induced quiescence, anoxic preconditioning, and embryonic diapause in embryos of the annual killifish Austrofundulus limnaeus

Embryos of the annual killifish Austrofundulus limnaeus can enter into dormancy associated with diapause and anoxia-induced quiescence. Dormant embryos are composed primarily of cells arrested in the G(1)/G(0) phase of the cell cycle based on flow cytometry analysis of DNA content. In fact, most cells in developing embryos contain only a diploid complement of DNA, with very few cells found in the S, G(2), or M phases of the cell cycle. Diapause II embryos appear to be in a G(0)-like state with low levels of cyclin D1 and p53. However, the active form of pAKT is high during diapause II. Exposure to anoxia causes an increase in cyclin D1 and p53 expression in diapause II embryos, suggesting a possible re-entry into the cell cycle. Post-diapause II embryos exposed to anoxia or anoxic preconditioning have stable levels of cyclin D1 and stable or reduced levels of p53. The amount of pAKT is severely reduced in 12?dpd embryos exposed to anoxia or anoxic preconditioning. This study is the first to evaluate cell cycle control in embryos of A. limnaeus during embryonic diapause and in response to anoxia and builds a foundation for future research on the role of cell cycle arrest in supporting vertebrate dormancy.     

Mouse oocytes gradually develop the capacity for activation during the metaphase II arrest

Metaphase II (M II) mouse oocytes were subjected to a parthenogenetic stimulus (8% ethanol) or fertilized in vitro at various times following the extrusion of the first polar body. The oocytes progressively develop the ability for full activation. Their responsiveness to activation stimuli not only increases, but also changes qualitatively with time. Newly arrested oocytes do not respond at all; then, when the ability to undergo meiotic anaphase II first develops, the response is defective: following extrusion of the second polar body (II PB), the oocyte does not enter interphase but arrests again at metaphase (M III-arrest). Finally, oocytes gain the ability for full activation including the entry to interphase. Depending on the type of activating stimulus, oocytes exhibit the capacity for full activation at different ages. The oocyte arrest in M III is similar to M II and can be released by subsequent activation. Such oocytes undergo anaphase III, extrude a third polar body (III PB), and form an aneuploid female pronuclei.     

NPP-16/Nup50 Function and CDK-1 Inactivation Are Associated with Anoxia-induced Prophase Arrest in Caenorhabditis elegans

Oxygen, an essential nutrient, is sensed by a multiple of cellular pathways that facilitate the responses to and survival of oxygen deprivation. The Caenorhabditis elegans embryo exposed to severe oxygen deprivation (anoxia) enters a state of suspended animation in which cell cycle progression reversibly arrests at specific stages. The mechanisms regulating interphase, prophase, or metaphase arrest in response to anoxia are not completely understood. Characteristics of arrested prophase blastomeres and oocytes are the alignment of condensed chromosomes at the nuclear periphery and an arrest of nuclear envelope breakdown. Notably, anoxia-induced prophase arrest is suppressed in mutant embryos lacking nucleoporin NPP-16/NUP50 function, indicating that this nucleoporin plays an important role in prophase arrest in wild-type embryos. Although the inactive form of cyclin-dependent kinase (CDK-1) is detected in wild-type–arrested prophase blastomeres, the inactive state is not detected in the anoxia exposed npp-16 mutant. Furthermore, we found that CDK-1 localizes near chromosomes in anoxia-exposed embryos. These data support the notion that NPP-16 and CDK-1 function to arrest prophase blastomeres in C. elegans embryos. The anoxia-induced shift of cells from an actively dividing state to an arrested state reveals a previously uncharacterized prophase checkpoint in the C. elegans embryo.     

The dynamic provision of different energy substrates improves development of one-cell random-bred mouse embryos in vitro.

Preliminary observations showed that one-cell embryos from random-bred MF1 mice avoid cleavage arrest at the two-cell stage ('in vitro two-cell block') when cultured in modified M16 culture medium containing lactate and pyruvate but lacking glucose. The roles of lactate, pyruvate and glucose during preimplantation development of embryos from random-bred mice in vitro were therefore examined. When all three substrates were present continuously during culture, one-cell embryos arrested at the two- to four-cell stages. Improved development to the morula stage after 96 h in culture was obtained in media containing pyruvate alone, lactate and pyruvate, pyruvate and glucose, lactate pyruvate and glucose for the first 24 h, and medium containing lactate and pyruvate for the remaining 72 h. In a second experiment, embryos were cultured in medium containing pyruvate alone, lactate and pyruvate or pyruvate and glucose for the first 24 h, and lactate plus pyruvate medium for the second 24 h. Subsequent transfer to medium containing lactate, pyruvate and glucose supported the morula to blastocyst transition. These results show that developmental arrest in vitro can be overcome by changing the combination of energy substrates at different stages of preimplantation development.     

Radical solutions and cultural problems: Could free oxygen radicals be responsible for the impaired development of preimplantation mammalian embryos in vitro?

A major obstacel to the study of mammalian development, and to the practical application of knowledge gained from it in the clinic during therapeutic in vitro fertilisation and embryo transfer (IVF-ET), is the propensity of embryos to become retarded or arrested during their culture in vitro. The precise developmental cell cycle in which embryos arrest or delay is characteristic for the species and coincides with the earliest period of embryonic gene expression. Much evidence reviewed here implicates free oxygen radicals (FORs) in the process of arrest. Thus, studies on the development of mouse preimplantation embryos in vitro have shown that (i) FORs are elevated in vitro, but not in vivo, at the time at which embryos become arrested or delayed, (ii) systems for removing reactive oxygen species to limit the formation of hydroxy radicals are present, although they have not yet been assessed quantitatively and may differ qualitatively from those in adult cells, (iii) metabolic and possibly genetic adaptations to oxidative damage are evident, (iv) published procedures for overcoming in vitro arrest are explicable in terms of FOR-mediated damage or responses and (v) the arrest or delay of most embryos in vitro can be reduced or prevented experimentally by addition of metal chelators to limit hydroxy radical formation and lipid hydroperoxidation.     

Cell cycle regulation during development and dormancy in embryos of the annual killifish Austrofundulus limnaeus

Embryos of the annual killifish Austrofundulus limnaeus can enter into a state of metabolic dormancy, termed diapause, as a normal part of their development. In addition, these embryos can also survive for prolonged sojourns in the complete absence of oxygen. Dormant embryos support their metabolism using anaerobic metabolic pathways, regardless of oxygen availability. Dormancy in diapause is associated with high ATP and a positive cellular energy status, while anoxia causes a severe reduction in ATP content and large reductions in adenylate energy charge and ATP/ADP ratios. Most cells are arrested in the G₁/G₀ phase of the cell cycle during diapause and in response to oxygen deprivation. In this paper, we review what is known about the physiological and biochemical mechanisms that support metabolic dormancy in this species. We also highlight the great potential that this model holds for identifying novel therapies for human diseases such as heart attack, stroke and cancer.     

Cell cycle regulation during development and dormancy in embryos of the annual killifish Austrofundulus limnaeus

Embryos of the annual killifish Austrofundulus limnaeus can enter into a state of metabolic dormancy, termed diapause, as a normal part of their development. In addition, these embryos can also survive for prolonged sojourns in the complete absence of oxygen. Dormant embryos support their metabolism using anaerobic metabolic pathways, regardless of oxygen availability. Dormancy in diapause is associated with high ATP and a positive cellular energy status, while anoxia causes a severe reduction in ATP content and large reductions in adenylate energy charge and ATP/ADP ratios. Most cells are arrested in the G₁/G₀ phase of the cell cycle during diapause and in response to oxygen deprivation. In this paper, we review what is known about the physiological and biochemical mechanisms that support metabolic dormancy in this species. We also highlight the great potential that this model holds for identifying novel therapies for human diseases such as heart attack, stroke and cancer.     

Dynamics of morphofunctional organization and developmental potential of mouse embryos in the state of "two-cell block in vitro"

Vital observation in combination with electron microscopy and immunocytochemistry was used for studying structural organization and developmental potential of BALB/c mouse embryos after cleavage cessation at a two cell stage caused by the "two-cell block in vitro" phenomenon. Modification of structure and viability of embryos was followed for 2 days from the time of cleavage arrest. Several hours before cleavage arrest, changes in mitochondrian distribution were noticed in embryos, no other disturbances in structural organization of blastomeres being obvious. Embryos, whose development was arrested for 24 h, remained viable and demonstrated some morphological changes similar to those seen in normally developing embryos of the same age. Towards the end of a 48 h block period some embryos died, the surviving embryos remained morphologically intact and metabolically active. At the same time, the nuclei of the latter frequently displayed chromatin condensation near the nuclear membrane, which is similar to the pattern of chromatin reorganization in the nuclei of early apoptotic cells. Our results support a concept on the "two-cell block in vitro" phenomenon as a specific functional state of embryos, and well compare with data on a partial realization by blocked embryos of the developmental program.     

Modulation of cell cycle control during oocyte‐to‐embryo transitions

Ex ovo omnia—all animals come from eggs—this statement made in 1651 by the English physician William Harvey marks a seminal break with the doctrine that all essential characteristics of offspring are contributed by their fathers, while mothers contribute only a material substrate. More than 360 years later, we now have a comprehensive understanding of how haploid gametes are generated during meiosis to allow the formation of diploid offspring when sperm and egg cells fuse. In most species, immature oocytes are arrested in prophase I and this arrest is maintained for few days (fruit flies) or for decades (humans). After completion of the first meiotic division, most vertebrate eggs arrest again at metaphase of meiosis II. Upon fertilization, this second meiotic arrest point is released and embryos enter highly specialized early embryonic divisions. In this review, we discuss how the standard somatic cell cycle is modulated to meet the specific requirements of different developmental stages. Specifically, we focus on cell cycle regulation in mature vertebrate eggs arrested at metaphase II (MII‐arrest), the first mitotic cell cycle, and early embryonic divisions.     

Heightened efficacy of nitric oxide-based therapies in type II diabetes mellitus and metabolic syndrome

Type II diabetes mellitus (DM) and metabolic syndrome are associated with accelerated restenosis following vascular interventions due to neointimal hyperplasia. The efficacy of nitric oxide (NO)-based therapies is unknown in these environments. Therefore, the aim of this study is to examine the efficacy of NO in preventing neointimal hyperplasia in animal models of type II DM and metabolic syndrome and examine possible mechanisms for differences in outcomes. Aortic vascular smooth muscle cells (VSMC) were harvested from rodent models of type II DM (Zucker diabetic fatty), metabolic syndrome (obese Zucker), and their genetic control (lean Zucker). Interestingly, NO inhibited proliferation and induced G0/G1 cell cycle arrest to the greatest extent in VSMC from rodent models of metabolic syndrome and type II DM compared with controls. This heightened efficacy was associated with increased expression of cyclin-dependent kinase inhibitor p21, but not p27. Using the rat carotid artery injury model to assess the efficacy of NO in vivo, we found that the NO donor PROLI/NO inhibited neointimal hyperplasia to the greatest extent in type II DM rodents, followed by metabolic syndrome, then controls. Increased neointimal hyperplasia correlated with increased reactive oxygen species (ROS) production, as demonstrated by dihydroethidium staining, and NO inhibited this increase most in metabolic syndrome and DM. In conclusion, NO was surprisingly a more effective inhibitor of neointimal hyperplasia following arterial injury in type II DM and metabolic syndrome vs. control. This heightened efficacy may be secondary to greater inhibition of VSMC proliferation through cell cycle arrest and regulation of ROS expression, in addition to other possible unidentified mechanisms that deserve further exploration.     

p53 Mediates the accelerated onset of senescence of endothelial progenitor cells in diabetes

Adverse metabolic factors, including oxidized small and dense low density lipoprotein (ox-dmLDL) can contribute to the reduced number and the impaired functions of circulating endothelial progenitors (EPC) in diabetic patients. To elucidate the molecular mechanisms involved, EPC from normal donors were cultured in the presence of ox-dmLDL. Under these experimental conditions EPC undergo to senescent-like growth arrest. This effect is associated with Akt activation, p21 expression, p53 accumulation, and retinoblastoma protein dephosphorylation and with a reduced protective effect against oxidative damage. Moreover, depletion of endogenous p53 expression by small interfering RNA demonstrates that the integrity of this pathway is essential for senescence to occur. Activation of the Akt/p53/p21 signaling pathway and accelerated onset of senescence are also detectable in EPC from diabetic patients. Finally, diabetic EPC depleted of endogenous p53 do not undergo to senescence-growth arrest and acquire the ability to form tube-like structures in vitro. These observations identify the activation of the p53 signaling pathway as a crucial event that can contribute to the impaired neovascularization in diabetes.     

Hypoxia and nitric oxide induce a rapid, reversible cell cycle arrest of the Drosophila syncytial divisions

Cells can respond to reductions in oxygen (hypoxia) by metabolic adaptations, quiescence or cell death. The nuclear division cycles of syncytial stage Drosophila melanogaster embryos reversibly arrest upon hypoxia. We examined this rapid arrest in real time using a fusion of green fluorescent protein and histone 2A. In addition to an interphase arrest, mitosis was specifically blocked in metaphase, much like a checkpoint arrest. Nitric oxide, recently proposed as a hypoxia signal in Drosophila, induced a reversible arrest of the nuclear divisions comparable with that induced by hypoxia. Syncytial stage embryos die during prolonged hypoxia, whereas post-gastrulation embryos (cellularized) survive. We examined ATP levels and morphology of syncytial and cellularized embryos arrested by hypoxia, nitric oxide, or cyanide. Upon oxygen deprivation, the ATP levels declined only slightly in cellularized embryos and more substantially in syncytial embryos. Reversal of hypoxia restored ATP levels and relieved the cell cycle and developmental arrests. However, morphological abnormalities suggested that syncytial embryos suffered irreversible disruption of developmental programs. Our results suggest that nitric oxide plays a role in the response of the syncytial embryo to hypoxia but that it is not the sole mediator of these responses.     

Bcl-2 can promote p53-dependent senescence versus apoptosis without affecting the G1/S transition

With the aim to identify events involved in the determination of p53-dependent apoptosis versus growth arrest, we used rat embryo fibroblasts expressing a temperature-sensitive mutant (tsA58) of the SV40 large tumour antigen (LT). Heat-inactivation of LT leads to p53 activation and commitment to a senescent-like state (REtsA15 cell line) or apoptosis (REtsAF cell line). We report that senescence is associated with high levels of the anti-apoptotic Bcl-2 protein and a cell cycle arrest in G1 phase, whereas apoptosis is associated with low levels of Bcl-2 and a cell cycle arrest in G2 phase. Here we show that Bcl-2, which can inhibit apoptosis and proliferation, turns the apoptotic phenotype into a senescent-like phenotype in G2 phase. This result suggests that Bcl-2-dependent inhibition of apoptosis could be crucial for the commitment to replicative senescence, whereas its ability to inhibit G1 progression would not be required.     

Induction of a senescent-like phenotype does not confer the ability of bovine immortal cells to support the development of nuclear transfer embryos

Previously, we reported that cloned embryos derived from an immortalized bovine mammary epithelial cell line (MECL) failed to develop beyond 12- to 16-cell stage. To analyze whether induction of a senescent-like phenotype in MECL can improve their ability to support the development after transfer into enucleated oocytes, we treated MECL with DNA methylation inhibitor 5-aza-2-deoxycytidine (Aza-C), histone deacetylase inhibitors trichostatin A (TSA), sodium butyrate (NaBu), or 5-bromodeoxyuridine and used those cells for nuclear transfer. Primary bovine fetal fibroblasts (BFF) were used as control. All agents were capable to induce features of senescence including reduced cell proliferation, enlarged cell size with a considerable proportion of cells stained positive for acidic senescence-associated beta-galactosidase and G1/S cell cycle boundary arrest in MECL. Aza-C treatment induced genome demethylation. Acetylation of H3 and H4 was increased after TSA treatment in both MECL and BFF, whereas no obvious changes in global H3 or H4 acetylation were detected after NaBu treatment. Nuclear transfer experiments following diverse treatments demonstrated that the induced senescent-like phenotype of MECL did not confer their ability to support embryonic development, although 7.3% of reconstructed embryos derived from NaBu-treated cells developed to morula stage. Intriguingly, a much higher proportion of cloned embryos developed to blastocysts when using NaBu-treated BFF, compared with using untreated BFF (59% versus 26%). Our results suggest that the developmental failure of donor nuclei from bovine immortal cells could not be reversed by induction of senescent-like phenotype. The beneficial effect of NaBu on the developmental potential of cloned embryos reconstructed from BFF merits further studies.