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Carnosine
and
Cellular Senescence
What the life cycles of cells and proteins tell us about mortality
What
makes cells mortal? Research findings on cellular senescence may explain not
only the life span potential of cells, but also cancer and human mortality
Most cells
regenerate themselves by dividing to form a pair of new cells. In 1961,
scientist L. Hayflick discovered that cells eventually reach a limit beyond
which they cannot continue to divide (Hayflick L et al., 1961; Hayflick L,
1965). In a now-famous series of experiments, Hayflick demonstrated that
cultured human fibroblasts (connective tissue cells) can divide only about 60 to
80 times. When a cell reaches this “Hayflick Limit” it enters into a
twilight state called cellular senescence. Senescent cells are very much
alive—yet they are distorted in both form and function.
Cultures of senescent cells cannot be mistaken for younger cells, which are
uniform in appearance and line up in parallel arrays. By contrast, senescent
cells exhibit a grainy appearance and take on odd shapes and sizes. They lose
the ability to organize themselves in a regular pattern. These striking changes
are called the senescent phenotype. A dipeptide (chemical union of two amino
acids) called carnosine has been shown to rejuvenate cells displaying the
senescent phenotype, quickly restoring the juvenile phenotype (McFarland GA et
al., 1999; McFarland GA et al., 1994).
Senescent cells also behave in deviant ways. For example, senescent dermal
(skin) cells generate more metalloproteinase enzymes that break down proteins in
the surrounding extracellular matrix (the fabric that holds together cells,
lymph nodes and blood vessels). They also generate more of the proinflammatory
cytokine (hormone-like proteins involved in cellular signaling) interleukin 1-a.
Senescent endothelial cells, which line blood vessel walls, generate higher
levels of an adhesion molecule that contributes to atherosclerosis. By secreting
damaging molecules and cytokines, senescent cells can disrupt the surrounding
tissue microenvironment. Relatively few cells could in this way exert
far-reaching deleterious effects upon tissue integrity and organ function
(Campisi J, 1997).
Is cellular senescence then tantamount to aging? There are several lines of
evidence supporting this conclusion. Cells from older people senesce after only
a fraction of the cell divisions that fetal cells can undergo. Cells from
short-lived animal species senesce faster than cells from long-lived species.
Cells from people with genetic premature aging syndromes senesce prematurely,
suggesting that the same genes regulate life span in the cell and the organism.
Finally, senescent cells accumulate with age in organs and tissues, where they
resist programmed cell death (apoptosis) and contribute to age-related
degeneration (Campisi J, 1997).
The cancer
connection
There is another direct connection between cellular senescence, aging and
mortality. Surprisingly, cellular senescence appears to be controlled by tumor
suppressor genes, including p53 and Rb (Bringold F et al., 2000; Campisi J,
2000, 1997). Most tumors contain cells that continue to divide beyond normal
limits or indefinitely. Tumor suppressor genes are thought to act in part by
inducing cellular senescence, which puts a halt to cell division. This has led
scientists to the intriguing hypothesis that cellular senescence evolved as a
defense against cancer. In support of this theory, recent research shows that
cells can respond to carcinogenic stimuli such as DNA damage and the activation
of cancer-promoting genes by entering a senescent state.
The
double-edged sword of cellular senescence thereby consigns cells to mortality
in order to protect them against cancer. Ironically, cellular senescence
alters the microenvironment around the cell in two ways that are thought to
contribute to both aging and carcinogenesis. First, senescent cells may impair
the structural integrity of the microenvironment, allowing a cell harboring an
oncogenic mutation to proliferate. For example, the enzymes secreted by
senescent dermal fibroblasts may be able to destroy the basement membrane and
underlying stroma (the tissue framework for an organ) that keep potentially
cancerous cells in check. Second, senescent cells overproduce growth factors
and cytokines that could stimulate the growth of precancerous cells. These
derangements of the structure and function of the cellular microenvironment
could synergize with accumulating mutations to favor the early stages of
tumorigenesis (Campisi J, 2000; Campisi J, 1997).
In addition, disturbances in cell cycle control due to inefficient protein
removal can set the stage for cancer, as we shall see below.
The protein
life cycle
The body is made up largely of proteins. The health of the body's stock of
proteins depends upon its freedom from damage (as through oxidation or
cross-linking), and upon its timely removal as part of normal protein
turnover.
The body's antioxidant system and other lines of defense cannot completely
protect proteins. Nature's second line of defense is the body's system for
repairing or removing damaged proteins. While some protein repair mechanisms
exist, there are no known ways to repair most protein damage, including even
simple oxidative damage to the amino acids which are the building blocks of
proteins. Thus it is essential for the body to efficiently remove aberrant and
unneeded proteins, a process called proteolysis.
Timely proteolysis removes damaged proteins before they do significant harm,
and removes undamaged proteins before they become damaged or disruptive. For
example, if oxidized proteins are not broken down, they tend to cross-link and
aggregate (as, for example, in cataracts or senile plaques). Rapid effective
proteolysis is therefore an anti-aging mechanism (Grune T et al., 1997).
The main proteolytic enzyme complex is called the proteasome. It removes
proteins that have been tagged for degradation by a peptide called ubiquitin.
Through its role in protein disposal, the proteasome-ubiquitin pathway helps
regulate many basic cellular processes including the cell cycle and cell
division, cell differentiation, cellular signaling, cellular metabolism and
DNA repair (Ciechanover A, 1998). Thus a malfunctioning proteasomal system has
far-reaching consequences.
As cells age, after many cell divisions, proteasome activity declines (Sitte N
et al., 2000; Merker K et al., 2000). At the same time, more and more proteins
undergo damage through a process called carbonylation. Thus the proteolytic
system becomes increasingly inadequate to deal with the increasing numbers of
abnormal or unneeded proteins, which can irreversibly form cross-links and
turn cellular processes awry.
New research shows that when the population of carbonylated proteins
permanently increases—as in aging—proteasome activity is depressed
(Petropoulos I et al., 2000; Keller JN et al., 2000; Burcham PC et al., 1997).
A vicious circle develops of age-related decline in proteasomal activity,
age-related increase in protein carbonylation and further inhibition of the
proteasome. The life cycles of proteins become blocked, and the normal
turnover of protein declines.
Is there a way to block this vicious circle? The body contains a dipeptide
called carnosine that both protects proteins from carbonylation and helps
reverse proteasomal decline. As in the aging body, proteolysis declines in
cultured cells as they approach senescence. Australian scientists showed that
carnosine enhances intracellular proteolytic activity in human connective
tissue cells (Hipkiss AR et al., 1995). Carnosine enhanced proteolysis the
most in old cells, and to a lesser extent in “middle aged” cells,
compensating for age-related proteolytic decline
The
cell cycle
The
concept of protein removal brings to mind structural proteins such as collagen
that the body breaks down after a relatively long life. In order to understand
the implications of proteolytic decline and buildup of aberrant proteins, it is
necessary to revise this picture.
Think
instead of a highly dynamic population of diverse proteins playing critical
roles in the body's regulatory and signaling pathways. These proteins must be
selectively synthesized at just the right moment so that they can do their
precisely timed jobs, then they must be swiftly degraded at the correct point in
a tightly regulated sequence of events. Normally such processes run like
clockwork, but when damaged proteins accumulate the system can bog down.
It
is in this way that physiological processes—both fast and slow—could become
deranged by excessive buildup of proteins marked for removal by the
proteasome-ubiquitin system. A case in point is the cell cycle. It consists of
four phases culminating in mitosis (cell division). The key steps in this cycle
are controlled by proteasomal degradation of proteins. For example, entry into
the DNA synthesis phase, separation of sister chromatids, and the exit from
mitosis are all dependent upon the timely removal of proteins such as cyclins by
the proteasome (Hershko A, 1997; Ford HL et al., 1999).
By
inhibiting the proteasome, carbonylated proteins could interfere with cell cycle
progression and control. To understand how this can happen, consider an engine
whose oil isn't changed regularly. When the detergent in the oil is used up,
contaminants precipitate and sludge forms on vital engine parts. The sludge
accumulates, impairing engine performance, until finally the engine dies.
The
body too needs an efficient sludge removal system. When protein “sludge”
accumulates, the gears of the cell cycle can get clogged up. This could impair
the efficiency of cell division, and perhaps more importantly, enable damaged
cells to reproduce. The result is increasing chromosomal instability, leading to
degeneration and cancer (Schmutte C et al., 1999). Another possible outcome is
cellular senescence, when the cell cycle grinds to a halt. Protein carbonylation
thus becomes a potentially terminal condition.
Cell cycle control represents one more pathway along which
damage to, and inefficient removal of protein could contribute to both cellular
mortality and the cellular immortalization seen in cancer. In this scenario, the
buildup of carbonylated protein feeds a vicious circle of proteasomal
impairment, chromosomal instability, and increasing numbers of defective and
senescent cells, which the body cannot remove. Insofar as the cellular life
cycle is bound up with the life cycles of proteins, it behooves us to maintain
healthy intact proteins and to ensure their timely turnover.
Skin
Aging
The
aging processes discussed above—cellular senescence, protein carbonylation and
proteasomal decline—play leading roles in the changes that aging brings to the
skin. While the epidermis (outer skin layer) changes only subtly with age,
profound changes take place in the dermis (inner skin layer). There, the
population of fibroblasts (connective tissue cells) is cut in half by age 80.
Collagen becomes disorganized with broken fibers, while the extracellular matrix
shows widespread destruction (West MD, 1994).
Protein
carbonylation damages all components of the epidermis and dermis, leading to
loss of elasticity, wrinkles, macromolecular disorganization, loss of
extracellular matrix and reduced capacity for wound repair—all of which are
primary characteristics of aged skin. Protein carbonylation rises with age in
the epidermis and in cultured keratinocytes (dividing cells that migrate into
the epidermis). As elsewhere in the body, it results from protein oxidation,
glycation (protein-sugar reactions) and reactions with lipid peroxidation
products (Petropoulos I et al., 2000).
Collagen,
the protein substance of connective tissue, tends to cross-link with age. It is
well known that collagen is cross-linked in the course of glycation and the
consequent formation of advanced glycation end products (AGEs). This robs the
skin of elasticity and youthful tone. Recent laboratory research demonstrates
that this problem can be self perpetuating. Once AGEs form, they can directly
induce the cross-linking of collagen—even in the absence of glucose and
oxidation reactions (Sajithlal GB et al., 1998). The researchers found that
neither antioxidants nor metal chelators could inhibit direct cross-linking of
collagen by AGEs. Only an anti-glycating agent, in this case the drug
aminoguanidine, could inhibit this process. The natural dipeptide carnosine
offers a superior efficacy and toxicity profile compared to aminoguanidine
(Munch G et al., 1997; Preston JE et al., 1998; Burcham PC, 2000).
The
researchers propose that reactive carbonyl compounds have the ability to induce
collagen cross-linking regardless of oxidative conditions. Their findings
underline the importance of preventing protein carbonylation and in particular
glycation before the cycle of collagen cross-linking gets started.
The
dynamic fibroblast
Connective
tissue cells, called fibroblasts, play the leading role in the ongoing
regeneration of the dermis. In order to function properly, fibroblasts must
strike a delicate balance between destruction of extracellular protein and
synthesis of new protein.
Normally
fibroblasts are quiescent, dividing at a low rate. They produce only small
amounts of the matrix metalloproteinase enzymes (collagenase and stromelysin)
that break down the surrounding extracellular matrix, and large amounts of
matrix metalloproteinase inhibitors (TIMP-1 and TIMP-2). But in response to
various stimuli including wounding and inflammation, they undergo a drastic
transformation into activated fibroblasts. They then secrete large amounts of
enzymes that break down collagen and destroy the extracellular matrix.
Cellular
senescence locks fibroblasts and keratinocytes into an approximation of this
activated state (West MD, 1994). They switch from a matrix-producing to a
matrix-degrading mode, secreting more matrix metalloproteinases and less matrix
metalloproteinase inhibitors. Senescent fibroblasts and keratinocytes are known
to accumulate in aging skin, as demonstrated by a biomarker of skin cell
senescence (Dimri GP et al., 1995). In addition to breaking down the
extracellular matrix, they secrete proinflammatory mediators such as
interleukin-1 alpha and growth factors such as heregulin (regulator of breast
and epithelial cell growth) whose influence extends far beyond the cell
secreting them (Campisi J, 1998; Campisi J, 1997).
Proteolysis
of connective tissue is a normal part of skin cell development and wound
healing. Proteolytic enzymes and their inhibitors sculpt structural proteins and
break them down at the appropriate times. Unfortunately, as aging skin cells
senesce and increase their proteolytic activity, the proteasome (the main enzyme
complex for protein degradation) enters an age-related decline. The balance
between protein creation and destruction is again upset, compromising the
integrity and regeneration of skin tissue.
New
research confirms that proteasome activity declines in keratinocytes and
epidermal cells with age. At the same time, protein carbonyl levels are rising
and the increasing numbers of senescent cells are secreting more proteolytic
enzymes. This has been demonstrated clearly in keratinocytes, both in culture
and in specimens from humans, where there is an inverse relationship between
proteasome content and biomarkers of cellular senescence (Petropoulos I et al.,
2000).
The skin makes visible the changes that occur throughout the body as damaged proteins and senescent cells accumulate. As we have seen, the life cycles of cells and proteins may regulate both our appearance as we age and how long we live. Preserving the integrity and regular turnover of protein is thus a key defense against the downward spirals of degeneration in the later years. Carnosine is the only agent that has shown multi-modal protective effects against protein degradation and cellular senescence.
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