Nicolle Sitte

Accumulation of single-strand breaks is the major cause of telomere shortening in human fibroblasts.

Telomere shortening triggers replicative senescence in human fibroblasts. The inability of DNA polymerases to replicate a linear DNA molecule completely (the end replication problem) is one cause of telomere shortening. Other possible causes are the formation of single-stranded overhangs at the end of telomeres and the preferential vulnerability of telomeres to oxidative stress. To elucidate the relative importance of these possibilities, amount and distribution of telomeric single-strand breaks, length of the G-rich overhang, and telomere shortening rate in human MRC-5 fibroblasts were measured. Treatment of nonproliferating cells with hydrogen peroxide increases the sensitivity to S1 nuclease in telomeres preferentially and accelerates their shortening by a corresponding amount as soon as the cells proliferate. A reduction of the activity of intracellular peroxides using the spin trap alpha-phenyl-t-butyl-nitrone reduces the telomere shortening rate and increases the replicative life span. The length of the telomeric single-stranded overhang is independent of DNA damaging stresses, but single-strand breaks accumulate randomly all along the telomere after alkylation. The telomere shortening rate and the rate of replicative aging can be either accelerated or decelerated by a modification of the amount of oxidative stress. Quantitatively, stress-mediated telomere damage contributes most to telomere shortening under standard conditions.

Proteasome inhibition by lipofuscin/ceroid during postmitotic aging of fibroblasts

We have studied the effects of hyperoxia and of cell loading with artificial lipofuscin or ceroid pigment on the postmitotic aging of human lung fibroblast cell cultures. Normobaric hyperoxia (40% oxygen) caused an irreversible senescence‐like growth arrest after about 4 wk and shortened postmitotic life span from 1–1/2 years down to 3 months. During the first 8 wk of hyperoxia‐induced ‘aging’, overall protein degradation (breakdown of [35S]methionine metabolically radiolabeled cell proteins) increased somewhat, but by 12 wk and thereafter overall proteolysis was significantly depressed. In contrast, protein synthesis rates were unaffected by 12 wk of hyperoxia. Lysosomal cathepsin‐specific activity (using the fluorogenic substrate z‐FR‐MCA) and cytoplasmic proteasome‐specific activity (measured with suc‐LLVY‐MCA) both declined by 80% or more over 12 wk. Hyperoxia also caused a remarkable increase in lipofuscin/ceroid formation and accumulation over 12 wk, as judged by both fluorescence measurements and FACscan methods. To test whether the association between lipofuscin/ceroid accumulation and decreased proteolysis might be causal, we next exposed cells to lipofuscin/ceroid loading under normoxic conditions. Lipofuscin/ceroid‐loaded cells indeed exhibited a gradual decrease in overall protein degradation over 4 wk of treatment, whereas protein synthesis was unaffected. Proteasome specific activity decreased by 25% over this period, which is important since proteasome is normally responsible for degrading oxidized cell proteins. In contrast, an apparent increase in lysosomal cathepsin activity was actually caused by a large increase in the number of lysosomes per cell. To test whether lipofuscin/ceroid could in fact directly inhibit proteasome activity, thus causing oxidized proteins to accumulate, we incubated purified proteasome with lipofuscin/ceroid preparations in vitro. We found that proteasome is directly inhibited by lipofuscin/ceroid. Our results indicate that an accumulation of oxidized proteins (and lipids) such as lipofuscin/ceroid may actually cause further increases in damage accumulation during aging by inhibiting the proteasome.–Sitte, N., Huber, M., Grune, T., Ladhoff, A., Doecke, W.‐D., von Zglinicki, T., Davies, K. J. A. Proteasome inhibition by lipofuscin/ceroid during postmitotic aging of fibroblasts. FASEB J. 14, 1490–1498 (2000)

Peroxynitrite Increases the Degradation of Aconitase and Other Cellular Proteins by Proteasome

We report that exposure of aconitase to moderate concentrations of peroxynitrite, 3-morpholinosydnonimine (SIN-1; a superoxide- and nitric oxide-liberating substance), or hydrogen peroxide, inhibits the enzyme and enhances susceptibility to proteolytic digestion by the isolated 20 S proteasome. Exposure to more severe levels of oxidative stress, from these same agents, causes further inhibition of the enzymatic activity of aconitase but actually decreases its proteolytic breakdown by proteasome. It should be noted that the superoxide and nitric oxide liberated by SIN-1 decomposition react to form a steady flux of peroxynitrite.S-Nitroso-N-acetylpenicillamine, a compound that liberates nitric oxide alone, causes only a small loss of aconitase activity (25% or less) and has no effect on the proteolytic susceptibility of the enzyme. Proteasome also seems to be the main protease in cell lysates that can degrade aconitase after it has been oxidatively modified by exposure to peroxynitrite, SIN-1, or hydrogen peroxide. Using cell lysates isolated from K562 cells treated for several days with an antisense oligodeoxynucleotide to the initiation codon region of the C2 subunit of proteasome (a treatment which diminishes proteasome activity by 50–60%), the enhanced degradation of moderately damaged aconitase was essentially abolished. Other model proteins as well as complex mixtures of proteins, such as cell lysates, also exhibit enhanced proteolytic susceptibility after moderate SIN-1 treatment. Therefore we conclude that peroxynitrite reacts readily with proteins and that mild modification by peroxynitrite results in selective recognition and degradation by proteasome.

Protein oxidation and degradation during cellular senescence of human BJ fibroblasts: part I—effects of proliferative senescence

Oxidized and cross‐linked proteins tend to accumulate in aging cells. Declining activity of proteolytic enzymes, particularly the proteasome, has been proposed as a possible explanation for this phenomenon, and direct inhibition of the proteasome by oxidized and cross‐linked proteins has been demonstrated in vitro. We have further examined this hypothesis during both proliferative senescence (this paper) and postmitotic senescence (see the accompanying paper, ref 1) of human BJ fibroblasts. During proliferative senescence, we found a marked decline in all proteasome activities (trypsin‐like activity, chymotrypsin‐like activity, and peptidyl‐glutamylhydrolyzing activity) and in lysosomal cathepsin activity. Despite the loss of proteasome activity, there was no concomitant change in cellular levels of actual proteasome protein (immunoassays) or in the steady‐state levels of mRNAs for essential proteasome subunits. The decline in proteasome activities and lysosomal cathepsin activities was accompanied by dramatic increases in the accumulation of oxidized and cross‐linked proteins. Furthermore, as proliferation stage increased, cells exhibited a decreasing ability to degrade the oxidatively damaged proteins generated by an acute, experimentally applied oxidative stress. Thus, oxidized and cross linked proteins accumulated rapidly in cells of higher proliferation stages. Our data are consistent with the hypothesis that proteasome is progressively inhibited by small accumulations of oxidized and cross‐linked proteins during proliferative senescence until late proliferation stages, when so much proteasome activity has been lost that oxidized proteins accumulate at ever‐increasing rates. Lysosomes attempt to deal with the accumulating oxidized and cross‐linked proteins, but declining lysosomal cathepsin activity apparently limits their effectiveness. This hypothesis, which may explain the progressive intracellular accumulation of oxidized and cross linked proteins in aging, is further explored during postmitotic senescence in the accompanying paper (1).—Sitte, N., Merker, K., von Zglinicki, T., Grune, T., Davies, K. J. A. Protein oxidation and degradation during cellular senescence of human BJ fibroblasts: part I—effects of proliferative senescence. FASEB J. 14, 2495–2502 (2000)

Proteasome‐dependent degradation of oxidized proteins in MRC‐5 fibroblasts

Fibroblasts were exposed to various concentrations of hydrogen peroxide and the removal of oxidized proteins was followed by determining protein‐bound carbonyls. Fibroblasts are able to increase the turnover of metabolically radiolabeled proteins after treatment with hydrogen peroxide. It was demonstrated for the first time, that the increased protein turnover was accompanied by a removal of protein‐bound carbonyl groups. The proteasome‐specific inhibitor lactacystin was able to inhibit the elimination of protein‐bound carbonyl groups. Therefore, the key role of the proteasome in the degradation of oxidized proteins in fibroblasts could be demonstrated.

β-Endorphin-containing memory-cells and μ-opioid receptors undergo transport to peripheral inflamed tissue

Abstract Immunocyte-derived β-endorphin can activate peripheral opioid receptors on sensory neurons to inhibit pain within inflamed tissue. This study examined μ-opioid receptors (MOR) on sensory nerves and β-endorphin (END) in activated/memory CD4 + cells (the predominant population homing to inflamed tissue). We found an upregulation of MOR in dorsal root ganglia, an increased axonal transport of MOR in the sciatic nerve and an accumulation of MOR in peripheral nerve terminals in Freunds adjuvant-induced hindpaw inflammation. A large number of CD4 + cells containing β-endorphin, but very few naive cells (CD45RC + ), were observed in inflamed tissue, suggesting that this opioid is mainly present in activated/memory cells (CD4 + /CD45RC − ). Taken together, our results indicate an enhanced transport of both MOR and of the endogenous ligand β-endorphin to injured tissue. This unique simultaneous upregulation of both receptors and ligands may serve to prevent excessive and/or chronic inflammatory pain.

Telomere shortening triggers a p53-dependent cell cycle arrest via accumulation of G-rich single stranded DNA fragments

It has been repeatedly suspected that telomere shortening might be one possible trigger of the p53-dependent cell cycle arrest, although the mechanism of this arrest remained unclear. Telomeres in human cells under mild oxidative stress accumulate single-strand damage faster than interstitial repetitive sequences. In MRC-5 fibroblasts and U87 glioblastoma cells, which both express wild-type p53, oxidative stress-mediated production of single-strand damage in telomeres is concomitant to the accumulation of p53 and p21 and to cell cycle arrest. This response can be modeled by treatment of cells with short single stranded telomeric G-rich DNA fragments. The arrest is transient in U87 cells. Recovery from it is accompanied by up-regulation of telomerase activity and elongation of telomeres. Overexpression of mutated p53 is sufficient to reverse the phenotype of inhibition as well as the delayed activation of telomerase. These data suggest that the production of G-rich single stranded fragments during the course of telomere shortening is sufficient to trigger a p53 dependent cell cycle arrest.

Protein oxidation and degradation during cellular senescence of human BJ fibroblasts: part II—aging of nondividing cells

Oxidized/cross‐linked intracellular protein materials, known as ceroid pigment, age pigment, or lipofuscin, accumulate in postmitotic tissues. It is unclear, however, whether diminishing proteolytic capacities play a role in the accumulation of such oxidized intracellular proteins. Previous studies revealed that the proteasome is responsible for the degradation of most oxidized soluble cytoplasmic and nuclear proteins and, we propose, for the prevention of such damage accumulations. The present investigation was undertaken to test the changes in protein turnover, proteasome activity, lysosome activity, and protein oxidation status during the aging of nondividing cells. Since the companion paper shows that both proteasome activity and the overall protein turnover decline during proliferative senescence whereas the accumulation of oxidized proteins increases significantly, we decided to use the same human BJ fibroblasts, this time at confluency, at different PD levels (including those that are essentially postmitotic) to investigate the same parameters under conditions where cells do not divide. We find that the activity of the cytosolic proteasome declines dramatically during senescence of nondividing BJ fibroblasts. The peptidyl‐glutamyl‐hydrolyzing activity was particularly affected. This decline in proteasome activity was accompanied by a decrease in the overall turnover of short‐lived (radiolabeled) proteins in the nondividing BJ fibroblasts. On the other hand, no decrease in the actual cellular proteasome content, as judged by immunoblots, was found. The decline in the activity of the proteasome was also accompanied by an increased accumulation of oxidized proteins, especially of oxidized and cross‐linked material. Unlike the loss of lysosomal function seen in our accompanying studies of proliferative senescence (1), however, the present study of hyperoxic senescence in nondividing cells actually revealed marked increases in lysosomal cathepsin activity in all but the very ‘oldest’ postmitotic cells. Our comparative studies of proliferating (1)and nonproliferating (this paper) human BJ fibroblasts reveal a good correlation between the accumulation of oxidized/cross‐linked proteins and the decline in proteasome activity and overall cellular protein turnover during in vitro senescence, which may predict a causal relationship during actual cellular aging.—Sitte, N., Merker, K., von Zglinicki, T., Davies, K. J. A., Grune, T. Protein oxidation and degradation during cellular senescence of human BJ fibroblasts: part II—aging of nondividing cells. FASEB J. 14, 2503–2510 (2000)

ACCELERATED TELOMERE SHORTENING IN FIBROBLASTS AFTER EXTENDED PERIODS OF CONFLUENCY

Telomere length in MRC-5 fibroblasts remains constant if the cells are proliferation-inhibited for up to 3 months by confluency. However, the apparent frequency of single-stranded sites in telomeres, measured as sensitivity to degradation by S1 nuclease, increases about fourfold during this extended inhibition of proliferation. After release of the cells, the frequency of telomeric single-stranded sites decreases to control values, and the telomere shortening rate increases about threefold as compared to controls proliferating without inhibition. This acceleration is transitory, the telomere shortening rate decreases to control values after about two population doublings after release. Finally, temporarily arrested fibroblast populations senesce at a lower cumulative population doubling level, but at about the same telomere length, as continuously proliferating controls. The data suggest that metabolic time-dependent single-strand degradation is a major cause of telomere shortening. They support the idea that telomere shortening plays an important role in triggering cellular senescence.

Protein oxidation and degradation during proliferative senescence of human MRC-5 fibroblasts

One of the highlights of age-related changes of cellular metabolism is the accumulation of oxidized proteins. The aging process on a cellular level can be treated either as the ongoing proliferation until a certain number of cell divisions is reached (the Hayflick limit) or as the aging of nondividing cells, that is, the age-related changes in cells without proliferation. The present investigation was undertaken to reveal the changes in protein turnover, proteasome activity, and protein oxidation status during proliferative senescence. We were able to demonstrate that the activity of the cytosolic proteasomal system declines dramatically during the proliferative senescence of human MRC-5 fibroblasts. Regardless of the loss in activity, it could be demonstrated that there are no changes in the transcription and translation of proteasomal subunits. This decline in proteasome activity was accompanied by an increased concentration of oxidized proteins. Cells at higher proliferation stages were no longer able to respond with increased degradation of endogenous [(35)S]-Met-radiolabeled proteins after hydrogen peroxide- or quinone-induced oxidative stress. It could be demonstrated that oxidized proteins in senescent human MRC-5 fibroblasts are not as quickly removed as they are in young cells. Therefore, our study demonstrates that the accumulation of oxidized proteins and decline in protein turnover and activity of the proteasomal system are not only a process of postmitotic aging but also occur during proliferative senescence and result in an increased half-life of oxidized proteins.