Cellular aging can be studied at two different levels: either as proliferative senescence, i.e., the loss of reproductive ability of a nontransformed cell culture; or as aging, and finally death, of individual (postmitotic) cells.
Major discoveries in the field of proliferative senescence have included the finding that telomere shortening may act as a mitotic clock and that cell cycle checkpoint control may be effected by the tumor suppressors p53 and pRB.
The finding that telomere shortening can be induced by oxidative stress recently connected this area of study with the free radical theory of aging.
Scientific understanding of the aging of individual postmitotic cells is much less advanced, although many theories have been proposed.
One such postulate is that the accumulation of heavily damaged, oxidized, and cross-linked proteins may contribute to the aging process in postmitotic cells.
Numerous studies have identified a major role for the 20S proteasome in the removal of oxidatively modified proteins in mammalian cells, and proteasome depletion prevents cells from degrading oxidized proteins.
Oxidized protein aggregates can inhibit the proteolytic activity of the proteasome in vitro.
Therefore, the accumulation of heavily damaged, oxidized, and aggregated proteins during postmitotic aging may diminish the effectiveness of proteolytic enzymes.
Lipofuscin and ceroid are fluorescent pigments of aggregated polymers derived from oxidation products of proteins and lipids, which are cross-linked by covalent and hydrophobic bonds.
Lipofuscin and ceroid accumulate during aging, most obviously in postmitotic cells.
A close correlation has been reported between lipofuscin/ceroid accumulation and aging rate in several mammalian species despite widely differing maximum life spans.
Lipofuscin/ceroid accumulation within aging cells might be due to increased production of reactive oxygen species or a decline in the efficiency of protein repair and/or degradation systems.
Experimentally, lipofuscin/ceroid accumulation can be accelerated by increased oxidative stress and by inhibition of lysosomal proteases and lipases, conditions that accelerate the aging process in general.
Accordingly, lipofuscin/ceroid accumulation is regarded as one of the best-known biomarkers of aging.
Since lipofuscin/ceroid is a biological marker of aging and cross-linked proteins are able to inhibit proteases, it has been decided to study the influence of oxidative stress and artificial lipofuscin/ceroid on protein turnover and proteolytic enzymes in aging postmitotic fibroblasts.
Scientists 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 weeks and shortened postmitotic life span from 1-½ years down to 3 months.
During the first 8 weeks of hyperoxia-induced "aging", overall protein degradation (breakdown of [35S] methionine metabolically radiolabeled cell proteins) increased somewhat, but by 12 weeks and thereafter overall proteolysis was significantly depressed.
In contrast, protein synthesis rates were unaffected by 12 weeks 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 weeks.
Hyperoxia also caused a remarkable increase in lipofuscin/ceroid formation and accumulation over 12 weeks, as judged by both fluorescence measurements and FACscan methods.
To test whether the association between lipofuscin/ceroid accumulation and decreased proteolysis might be causal, scientists 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 weeks 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.
It has been found that proteasome is directly inhibited by lipofuscin/ceroid.
These 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.