Replicative senescence: a critical review.

Human cells in culture have a limited proliferative capacity. After a period of vigorous proliferation, the rate of cell division declines and a number of changes occur in the cells including increases in size, in secondary lysosomes and residual bodies, nuclear changes and a number of changes in gene expression which provide biomarkers for senescence. Although human cells in culture have been used for over 40 years as models for understanding the cellular basis of aging, the relationship of replicative senescence to aging of the organism is still not clear. In this review, we discuss replicative senescence in the light of current information on signal transduction and mitogenesis, cell stress, apoptosis, telomere changes and finally we discuss replicative senescence as a model of aging in vivo.

Effects of oxygen on the antioxidant responses of normal and transformed cells.

Basal antioxidant defense levels are often aberrant in tumor cells; however, less attention has been given to differences in the way that normal and transformed cells respond to changes in oxidative stress. This study evaluated differences in the responses of various normal and transformed cell lines to different oxygen tensions. Exposure to hyperoxia generally failed to induce either the activity of GSH peroxidase (GPx) or the manganese-containing form of superoxide dismutase (MnSOD) after 48 h, although at 605 mm Hg oxygen, small inductions of MnSOD activity were observed in adult lung fibroblasts and amelanotic melanoma. Exposure to 605 mm Hg O2 for 48 h was inhibitory to GPx activity. MnSOD activity was strongly induced in virally transformed WI-38 cells by treatment with the herbicide paraquat or inhibition of GSH synthesis with BSO. In normal cells GSH concentration was proportional to ambient oxygen tension. Tumor cells exhibited greater GSH concentrations at low oxygen tensions than normal cells but were unable to increase GSH in response to elevation of oxygen tension. These results reveal differences in tumor and normal cell responses to changes in ambient oxygen tension and show that MnSOD activity is inducible when an appropriate stimulus is applied.

Regulation of peptidergic vesicle mobility by secretagogues.

Neuropeptides are released into the extracellular space from large secretory granules. In order to reach their release sites, these granules are translocated on microtubules and thought to interact with filamentous actin as they approach the cell membrane. We have used a green fluorescent protein-tagged neuropeptide prohormone (prepro-orphanin FQ) to visualize vesicle trafficking dynamics in NS20Y cells and cultures of primary hippocampal neurons. We found that the majority of secretory granules were mobile and accumulated at both the tips of neurites as well as other apparently specialized cellular sites. We also used live-cell imaging to test the notion that peptidergic vesicle mobility was regulated by secretagogues. We show that treatment with forskolin appeared to increase vesicle rates of speed, while depolarization with high K+ had no effect, even though both treatments stimulated neuropeptide secretion. In cultured hippocampal neurons the green fluorescent protein-tagged secretory vesicles were routed to both dendrites and axons, indicating that peptidergic vesicle transport was not polarized. Basal peptidergic vesicle mobility rates in hippocampal neurons were the same as those in NS20Y cells. Taken together, these studies suggest that secretory vesicle mobility is regulated by specific classes of secretagogues and that neuropeptide containing secretory vesicles may be released from dendritic structures.

Effects of establishing cell cultures and cell culture conditions on the proliferative life span of human fibroblasts isolated from different tissues and donors of different ages.

It is well known that normal human cells placed in a culture environment exhibit a limited proliferative capacity. The extent to which the culture environment influences proliferative life span is not understood. This study evaluated the effects of the standard procedures used to establish and maintain cultures on the proliferative life spans of different types of human fibroblast cells established from fetal and adult skin and lung. The results of this study demonstrate that procedures to establish cell cultures use only one of several subpopulations of cells present in biopsy pieces and that the culture conditions routinely employed by most laboratories can exert significant effects on proliferative life-span determinations. The maximum proliferative life span differed significantly when obtained by growing the cells in two commonly used commercial media. Proliferative life span was inversely related to ambient oxygen tension and directly related to seeding density in all of the lines examined although lines established from adult skin were much more resistant to toxicity. Enzymatic antioxidant defense levels of fetal skin fibroblasts were much lower than those observed in adult skin fibroblasts, but the effects of oxygen on their life spans were similar. Hyperoxia induced larger increases in glutathione concentration in cell lines with low antioxidant enzyme levels.

Effects of ambient oxygen concentration on the growth and antioxidant defenses of of human cell cultures established from fetal and postnatal skin.

Oxygen toxicity is believed to arise from changes in the rates at which cells generate reactive oxygen species (ROS). Sensitivity to hyperoxia has been postulated to depend on levels of antioxidant defense. Human cells obtained from fetal tissues have lower antioxidant defenses than those obtained from adult tissue. The present study was performed to determine whether the differences in fetal and adult antioxidant defense levels modulated their responses to changes in the ambient oxygen concentration. Our results demonstrate that oxygen modulates the proliferation of human fetal and adult skin fibroblasts in a similar fashion. In general, skin fibroblasts grew better at approximately 31 mm Hg, regardless of donor age. Manganese superoxide dismutase, catalase, and glutathione peroxidase activities were lower in fetal cells than in adult fibroblasts. Copper/zinc superoxide dismutase and glucose-6-phosphate dehydrogenase were similar in fetal and postnatal tissues and were unaltered appreciably by hyperoxic exposure. Glutathione concentration increased at higher oxygen tensions; however, the increase was much greater in fetal cells than in cultures derived from adult skin. These observations demonstrate that the capacity of fetal and adult cells to cope with oxidative stress, while similar, result from distinct mechanisms.

Mitochondrial DNA mutation analysis in human skin fibroblasts from fetal, young, and old donors.

Multibase deletions in mitochondrial DNA (mtDNA) have been shown to accumulate with age in several tissues, including skin, whereas point mutations have only recently been demonstrated to increase during aging, with several specific mutations occurring at high levels (up to 50%) in skin fibroblasts obtained from old donors [Science 286(1999)774]. We have conducted a survey for a specific deletion and for point mutations in several regions of mtDNA from cultured skin fibroblasts derived from eight fetal (12-20 weeks gestational age), ten young (17-33 years of age) and 11 old (78-92 years of age) human donors. Using PCR analysis, detectable levels of the 4977 basepair (bp) 'common deletion' were present in all three age groups, with the highest deletion levels of up to 0.3% of total mtDNA found in several cell lines from old donors, although other old donor cell lines had much lower levels. Single strand conformation polymorphism (SSCP) analysis for point mutations in the non-coding D-loop region and two regions of the cytochrome oxidase 2 gene failed to reveal the presence of any single base mutations. We infer that age-related high level mutational damage in mtDNA from human skin fibroblasts may manifest both sequence and inter-individual specificity.

Ablation of pituitary pro-opiomelanocortin (POMC) cells produces alterations in hypothalamic POMC mRNA levels and midbrain mu opioid receptor binding in a conditional transgenic mouse model.

The hypothalamic-pituitary-adrenal (HPA) axis is regulated by stress-related excitatory inputs, and various inhibitory and negative-feedback controls by glucocorticoids and opioids, including pro-opiomelanocortin (POMC)-derived peptides. The role of POMC-derived peptides of pituitary origin in the modulation of brain POMC mRNA expression and opioid receptor binding was investigated using a line of transgenic mice that express a fusion gene composed of the pituitary expression-specific promoter region of the POMC gene driving the herpes simplex viral-1 thymidine kinase (TK). Male adult mice were treated with the antiherpes agent ganciclovir that selectively ablates cells expressing TK. Following treatment, POMC mRNA levels, measured by quantitative solution hybridization/RNase protection assays, were decreased by 48% in the pituitary of the TK+/+ mice, reflecting an expected loss of the pituitary corticotrope POMC cells. This treatment also significantly lowered pituitary beta-endorphin immunoreactivity content and plasma concentrations of corticosterone. In contrast, POMC mRNA levels were increased by 79% in the hypothalamus of the TK+/+ mice with pituitary POMC cell ablation. Binding of [(3)H]DAMGO to mu opioid receptors, as measured by quantitative autoradiography, was significantly reduced in several brain regions including the central grey, median raphe and superficial grey layer of the superior colliculus. These regions are innervated by hypothalamic POMC neurones. No significant differences in binding to either kappa or delta opioid receptors were found in the brain regions studied. These results suggest that POMC-derived peptides of pituitary origin may exert a tonic negative-feedback effect on hypothalamic POMC neurones. In turn, the downregulation of central mu opioid receptors in this model may be mediated through a mechanism related to hypothalamic POMC overexpression.

Lack of Elk-1 phosphorylation and dysregulation of the extracellular regulated kinase signaling pathway in senescent human fibroblast.

Replicative senescence is characterized by numerous phenotypic alterations including the loss of proliferative capacity in response to mitogens and numerous changes in gene expression including impaired serum inducibility of the immediate-early genes c-fos and erg-1. Transcription of c-fos in response to mitogens depends on the activation of a multiprotein complex formed on the c-fos serum response element (SRE), which includes the transcription factors SRF (serum response factor) and TCF (ternary complex factor). Our data indicate that at least two defects are responsible for the decreased c-fos transcription in senescent cells, one caused by diminished DNA binding activity of the SRF and another resulting from impaired activation of the TCF, Elk-1. In nuclei isolated from serum stimulated senescent cells the activating phosphorylation of p62(TCF)/Elk-1, which is catalyzed by the members of the extracellular-regulated kinase (ERK) family was strikingly diminished and correlated with a decrease in the abundance of activated ERK proteins. In contrast, in total cell lysates ERK phosphorylation and ERK activity (normalized to total protein) reached similar levels following stimulation of early- and late-passage cells. Interestingly, senescent cells consistently exhibited higher ERK protein abundance. Thus, the proportion of phosphorylated (active) ERK molecules in stimulated senescent cells was lower than in early passage cells. The accumulation of unphosphorylated ERK molecules in senescent cells correlated with the diminished abundance of phosphorylated (active) MEK. These data indicate that in senescent cells there is a general dysregulation in the ERK signaling pathway, which results in the accumulation of inactive ERK molecules, decreased abundance of active ERK in the nucleus of senescent cells, and subsequent lack of activation of the transcription factor TCF(Elk-1). These impairments, together with the impaired DNA binding activity of SRF, could potentially account for the lack of c-fos expression in senescent cells and for multiple other molecular changes dependent upon this pathway.

Altered processing of pro-orphanin FQ/nociceptin and pro-opiomelanocortin-derived peptides in the brains of mice expressing defective prohormone convertase 2.

The bioactivity of neuropeptides can be regulated by a variety of post-translational modifications, including proteolytic processing. Here, gene-targeted mice producing defective prohormone convertase 2 (PC2) were used to examine the post-translational processing of two neuroendocrine prohormones, pro-opiomelanocortin (POMC) and pro-orphanin FQ (pOFQ)/nociceptin (N), in the brain. Reversed-phase HPLC and gel-exclusion chromatography were combined with specific radioimmunoassays to analyze the processing patterns of these two prohormones in the hypothalamus and the amygdala. In the case of POMC, the lack of PC2 activity completely prevented carboxy-shortening of beta-endorphins and greatly diminished conversion of beta-lipotropin to gamma-lipotropin and beta-endorphin. Although conversion of beta-lipotropin to beta-endorphin decreased, the lack of PC2 activity caused an increase in beta-lipotropin and beta-endorphin levels in the mutant animals, but no increases in POMC or biosynthetic intermediates were seen. The extent of OFQ/N production was significantly lower in PC2-deficient mice and there was an accumulation of relatively large amounts of pOFQ/N and biosynthetic intermediates. These results demonstrate that PC2 is directly involved in the biogenesis of two brain neuropeptides in vivo and suggest that the specific prohormone and cellular context influences neuropeptide processing by PCs.

Actions of nociceptin/orphanin FQ and other prepronociceptin products on rat rostral ventromedial medulla neurons in vitro.

1. Whole-cell patch clamp recordings were made from rat rostral ventromedial medulla (RVM) neurons in vitro to investigate the cellular actions of the opioid-like receptor ORL1 (NOP), ligand nociceptin/orphanin FQ and other putative prepronociceptin products. 2. Primary and secondary RVM neurons were identified as responding to the kappa-opioid receptor agonist U-69593 (300 nM to 1 microM) and the mu- and delta-opioid receptor agonist met-enkephalin (10 microM), respectively. Both primary and secondary RVM neurons responded to nociceptin (3 nM to 1 microM) with an outward current that reversed polarity at -115 mV in brain slices and with inhibition of Ca(2+) channel currents in acutely isolated cells. 3. The putative ORL1 antagonist J-113397 (1 microM) produced no change in membrane current and abolished the outward current produced by nociceptin (100 nM). In contrast, Phe(1)psi(CH(2)-NH)Gly(2)]-nociceptin-(1-13)NH(2) (300 nM to 1 microM) alone produced an outward current and partially reduced the outward current produced by nociceptin (300 nM) when co-applied. 4. In brain slices nociceptin (300 nM) reduced the amplitude of evoked GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSCs) but not non-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs). 5. Met-enkephalin (10 microM), but not nociceptin (300 nM), reduced the rate of spontaneous miniature IPSCs in normal external potassium solution (K(+) 2.5 mM). In high external potassium (K(+) 17.5 mM), nociceptin reduced the rate of miniature IPSCs in the presence (Ca(2+) 2.4 mM, Mg(2+) 1.2 mM) but not in the absence of external calcium (Ca(2+) 0 mM, Mg(2+) 10 mM, Cd(2+) 10 microM). Nociceptin and met-enkephalin had no effect on the amplitude of miniature IPSCs. 6. The putative nociceptin precursor products nocistatin (rat prepronociceptin(125-132)) and rat prepronociceptin(154-181) had no effect on membrane currents, evoked IPSCs and evoked EPSCs. 7. These results indicate that nociceptin acts via the ORL1 receptor to directly inhibit both primary and secondary RVM neurons by activating a potassium conductance and by inhibiting calcium conductances. In addition, nociceptin inhibits GABA release within the RVM via a presynaptic Ca(2+)-dependent mechanism. Thus, nociceptin has the potential to exert both disinhibitory and inhibitory effects on neuronal action potential firing within the RVM.

Matrix and cell injury due to sub-impact loading of adult bovine articular cartilage explants: effects of strain rate and peak stress.

Mechanical overloading of cartilage has been implicated in the initiation and progression of osteoarthrosis. Our objectives were to identify threshold levels of strain rate and peak stress at which sub-impact loads could induce cartilage matrix damage and chondrocyte injury in bovine osteochondral explants and to explore relationships between matrix damage, spatial patterns of cell injury, and applied loads. Single sub-impact loads characterized by a constant strain rate between 3 x 10(-5) and 0.7 s(-1) to a peak stress between 3.5 and 14 MPa were applied, after which explants were maintained in culture for four days. At the higher strain rates, matrix mechanical failure (tissue cracks) and cell deactivation were most severe near the cartilage superficial zone and were associated with sustained increased release of proteoglycan from explants. In contrast, low strain rate loading was associated with cell deactivation in the absence of visible matrix damage. Furthermore, cell activity and proteoglycan synthesis were suppressed throughout the cartilage depth, but in a radially dependent manner with the most severe effects at the center of cylindrical explants. Results highlight spatial patterns of matrix damage and cell injury which depend upon the nature of injurious loading applied. These patterns of injury may also differ in terms of their long-term implications for progression of degradative disease and possibilities for cartilage repair.

Carboxyl terminal peptides derived from prepro-orphanin FQ/nociceptin (ppOFQ/N) are produced in the hypothalamus and possess analgesic bioactivities.

Orphanin FQ/nociceptin (OFQ/N), the endogenous ligand for the ORL-1/KOR-3 receptor, produces a wide variety of behavioral responses. Its precursor protein, prepro-OFQ/N (ppOFQ/N) contains several series of amino acids bounded by pairs of basic amino acids, raising the possibility that additional functional neuropeptides could be generated by proteolytic posttranslational processing. Several of these processing products have been shown to have pharmacological activity, including the 17 amino acid peptide OFQ/N (mppOFQ/N(140-157)) which is a major product of this precursor in the hypothalamus. Here we have used a newly developed radioimmunoassay and RP-HPLC to detect mppOFQ/N(160-187) in mouse hypothalamic extracts. Murine ppOFQ/N(160-187) has potent analgesic activity supraspinally (3.4 nmol, i.c.v.) and spinally (4.3 nmol, i.t.). This analgesic activity was reversed by the opioid antagonist naloxone (5 mg/kg, s.c.) and kappa(1)-selective opioid antagonist nor-BNI (60 microg, i.c.v.), despite the inability of ppOFQ/N(160-187) to compete binding in mu, delta, kappa(1), kappa(3), or OFQ/N binding assays. These findings suggest that murine ppOFQ/N(160-187) may be a physiologically relevant neuropeptide with a novel mechanism of action.

Integrity of tritiated orphanin FQ/nociceptin: implications for establishing a reliable binding assay.

In the course of establishing a reliable and reproducible binding assay for the orphanin FQ/nociceptin (OFQ/N) ligand-receptor system we used reversed phase-high-performance liquid chromatography (HPLC) (RP-HPLC) to monitor the integrity of [(3)H]OFQ/N obtained from three different manufacturers. This means of analysis revealed that the stability of [(3)H]OFQ/N during storage varied considerably depending on the manufacturer. Furthermore, the integrity of [(3)H]OFQ/N was significantly compromised in the presence of COS-7 cell membranes. Interestingly, if the peptide was added to COS-7 membranes after they had been exposed to low pH it remained intact, suggesting that the peptide's breakdown during binding is, in part, enzymatically mediated. Although a variety of protease inhibitors were tested, none proved completely effective at protecting the tritiated peptide. The intention of the studies presented here was to evaluate OFQ/N binding components, namely the available [(3)H]OFQ/N ligands, in an effort to standardize the binding conditions for this receptor ligand system. Consequently, this study underscores the importance of monitoring the integrity of the trace ligand being used in a given binding assay.

Motilides accelerate regional gastrointestinal transit in the dog.

BACKGROUND: Motilides have prokinetic effects on the upper gut during fasting, increasing the strength of antral contractions and stimulating gastroduodenal phase 3 sequences. Effects on the distal gut, and postprandially, are less well documented. AIM: To evaluate dose-response effects of motilin and erythromycin on gastric emptying, small bowel and colonic transit in the dog using a validated scintigraphic technique. METHODS: For gastric emptying and small bowel transit, 99mTc labelled beads were added to a meal of dog chow (450 kcal). Regional colonic transit was measured by 111In labelled beads placed in a capsule which dissolved and released radiation into the proximal colon. Scintiscans were taken at regular intervals and indices of whole-gut transit were calculated. Drugs were given by slow intravenous administration. RESULTS: In the doses used, motilin accelerated regional colonic transit but did not hasten gastric emptying or small bowel transit. Single or repeated doses of motilin had similar effects on colonic transit. Erythromycin accelerated gastric emptying, small bowel transit and regional colonic transit. CONCLUSIONS: Motilin receptors are apparently present in the canine small bowel and colon. Postprandially, motilides accelerate transit in the distal gut.

The hemodynamic effects of prolonged respiratory alkalosis in anesthetized newborn piglets.

OBJECTIVE: To test the hypothesis that prolonged alkalosis decreases cardiac output and, furthermore, exacerbates hypoxic pulmonary vasoconstriction, as respiratory alkalosis is frequently induced as a therapy for persistent pulmonary hypertension of the newborn despite a lack of controlled evidence of improved outcomes. Potential adverse effects of prolonged alkalosis have been demonstrated. METHOD: Two groups (control, n = 6, and hypocapnic alkalosis, n = 6) of 1-3 day old fentanyl-anesthetized, vecuronium-paralyzed piglets were instrumented to measure cardiac index (CI) and mean systemic (MAP) and pulmonary (PAP) arterial pressures. Baseline values were recorded. Alveolar hypoxia was then induced to achieve an arterial oxygen saturation of between 50 and 60% for 15 min. Respiratory alkalosis was then induced, by increasing ventilation to achieve a pH between 7.55-7.60, and was continued for 240 min. Inspired carbon dioxide was used with hyperventilation in the control group to maintain pressure of arterial carbon dioxide (PaCO2) at 35-45 mmHg and pH of 7.35-7.45. Hypoxia was induced again at 15 and 240 min. Pulmonary and systemic vascular resistances (PVR and SVR) were calculated. RESULTS: Prolonged alkalosis led to a significant and progressive fall in mean MAP from 61 (SD 7) mmHg at the start of the study falling to 50 (SD 6.9, p = 0.043), with no effect on CI. Calculated SVR decreased (0.45 SD 0.03 vs 0.36 SD 0.05). There were no statistically significant changes in any of the variables in the control group. Neither acute nor prolonged respiratory alkalosis had a significant effect on hypoxic pulmonary vasoconstriction. CONCLUSIONS: Prolonged hyperventilation leads to systemic hypotension, however it does not exacerbate hypoxic pulmonary vasoconstriction.

Is beta-galactosidase staining a marker of senescence in vitro and in vivo?

Cytochemically detectable beta-galactosidase (beta-gal) at pH 6.0 has been reported to increase during the replicative senescence of fibroblast cultures and has been used widely as a marker of cellular senescence in vivo and in vitro. In this study, we have characterized changes in senescence-associated (SA) beta-gal staining in early and late passage cultures, cultures established from donors of different ages, virally immortalized cells, and tissue slices obtained from donors of different ages. The effects of different culture conditions were also examined. While we confirm the previous report that SA beta-gal staining increased in low-density cultures of proliferatively senescent cells, we were unable to demonstrate that it is a specific marker for aging in vitro. Cultures established from donors of different ages stained for SA beta-gal activity as a function of in vitro replicative age, not donor age. We also failed to observe any differences in SA beta-gal staining in skin cells in situ as a marker of aging in vivo. The level of cytochemically detectable SA beta-gal was elevated in confluent nontransformed fibroblast cultures, in immortal fibroblast cultures that had reached a high cell density, and in low-density, young, normal cultures oxidatively challenged by treatment with H2O2. Although we clearly demonstrate that SA beta-gal staining in cells is increased under a variety of different conditions, the interpretation of increased staining remains unclear, as does the question of whether the same mechanisms are responsible for the increased SA beta-gal staining observed in senescent cells and changes observed in cells under other conditions.

Oxidative stress and gene regulation.

Reactive oxygen species are produced by all aerobic cells and are widely believed to play a pivotal role in aging as well as a number of degenerative diseases. The consequences of the generation of oxidants in cells does not appear to be limited to promotion of deleterious effects. Alterations in oxidative metabolism have long been known to occur during differentiation and development. Experimental perturbations in cellular redox state have been shown to exert a strong impact on these processes. The discovery of specific genes and pathways affected by oxidants led to the hypothesis that reactive oxygen species serve as subcellular messengers in gene regulatory and signal transduction pathways. Additionally, antioxidants can activate numerous genes and pathways. The burgeoning growth in the number of pathways shown to be dependent on oxidation or antioxidation has accelerated during the last decade. In the discussion presented here, we provide a tabular summary of many of the redox effects on gene expression and signaling pathways that are currently known to exist.

Use of the fibroblast model in the study of cellular senescence.

In this chapter, we present standard procedures for the culture of human cells that exhibit a finite proliferative capacity (replicative life-span). The use of a cell culture model has the advantage of providing a controlled environment to study a wide variety of cellular phenomena. It also has the inherent limitation of isolating cells from the regulatory elements that might be provided by other types of cells in vivo. Nevertheless, cell culture models have been crucial to our current understanding of mechanisms of growth, differentiation, development, and neoplasia and numerous other disease states. In this chapter we present procedures for human fibroblast culture including serumfree cultivation of cells, which is necessary when the cellular environment must be fully defined. In addition, we present procedures for the determination of replicative life-span, saturation density, and assessment of replicative capacity from labeled thymidine incorporation in fibroblasts. The methods described here have been well tested and provide highly reproducible results (1, 2).