X-Message-Number: 25039
Date: Sun, 14 Nov 2004 12:31:15 -0800 (PST)
From: Doug Skrecky <>
Subject: Molecular Markers Of Aging Identified

CHAPEL HILL -- Researchers at the University of North Carolina at Chapel
Hill Lineberger Comprehensive Cancer Center may have made a crucial
discovery in the understanding of cellular aging.

In a study published in the Nov. 1 issue of the Journal of Clinical
Investigation, the researchers report that as cells and tissues age, the
expression of two proteins called p16INK4a and ARF dramatically
increases. This increase in expression, more than a hundredfold in some
tissues, suggests a strong link between cellular aging and the
upregulation, or increased production, of p16INK4a and ARF.

"At the very least, our work suggests that looking at the expression of
one or both proteins will make a great biomarker of aging - a tool to
clinically determine the actual molecular age of people, as opposed to
just their chronological age," said Lineberger member Dr. Norman
Sharpless, the senior author of the study and assistant professor of
medicine and genetics at UNC's School of Medicine.

"We all know people that we consider to be a young 65, and we believe
they won't demonstrate as much p16INK4a or ARF expression as others of
the same age."

In addition to identifying molecular targets that may slow aging in the
future, the study may also suggest immediate clinical applications.
Knowing the molecular age of a tissue may also enable physicians to
select the "youngest" most viable tissues and organs for transplantation,
to predict how well a patient will heal after surgery and, by being able
to characterize the regenerative ability of a patient's bone marrow,
predict future toxicity of chemotherapy in a cancer patient.

Both p16INK4a and ARF are known potent tumor suppressors, or proteins
that halt tumor cell growth. The study suggests that the important
anti-cancer function of these proteins to limit cellular growth might in
turn cause aging.

"Proliferation of cells is important in the repair and regrowth of
tissues. In fact, we grow old in part because our bodies' ability to
regenerate tissues decreases as we age," Sharpless said. "We believe an
untoward effect of increased p16INK4a and ARF expression outside of
cancer is a decrease in cellular proliferation needed to sustain this

The researchers also found that the increase in p16INK4a and ARF can be
substantially inhibited by decreasing caloric intake, a known retardant
of aging. This result suggests that decreased expression of p16INK4a and
ARF could mediate the known anti-aging effects of caloric restriction.

"Our results suggest that going on a short-term diet will not reverse the
aging process; only long-term restrictions appeared to have an effect on
p16INK4a and ARF expression. Therefore, our results would not be
consistent with the idea that short-term caloric restriction prior to
surgery would improve post-operative wound healing," Sharpless said.

The work has strong implications for stem cell renewal, he added. Stem
cells are self-replenishing cells that constantly divide and
differentiate into the component cells that make up the tissues in the
human body and are found in particularly high number in the bone marrow,
as well as organs such as the skin, kidney and liver.

"As tissue stem cells age, they appear to express more p16INK4a and ARF,
which would stop those cells from replenishing," Sharpless said. "As
people age, they could just run out of functioning stem cells."

Depletion of stem cells could affect the ability of the body to heal
after injury or surgery and may also predict the ability of certain
diseases, such as cardiomyopathy, to progress," he added.

J Clin Invest. 2004 Nov;114(9):1299-307
Ink4a/Arf expression is a biomarker of aging.
Krishnamurthy J, Torrice C, Ramsey MR, Kovalev GI, Al-Regaiey K, Su L,
Sharpless NE.
  The Ink4a/Arf locus encodes 2 tumor suppressor molecules, p16(INK4a) and
Arf, which are principal mediators of cellular senescence. To study the
links between senescence and aging in vivo, we examined Ink4a/Arf
expression in rodent models of aging. We show that expression of
p16(INK4a) and Arf markedly increases in almost all rodent tissues with
advancing age, while there is little or no change in the expression of
other related cell cycle inhibitors. The increase in expression is
restricted to well-defined compartments within each organ studied and
occurs in both epithelial and stromal cells of diverse lineages. The
age-associated increase in expression of p16(INK4a) and Arf is attenuated
in the kidney, ovary, and heart by caloric restriction, and this decrease
correlates with diminished expression of an in vivo marker of senescence,
as well as decreased pathology of those organs. Last, the age-related
increase in Ink4a/Arf expression can be independently attributed to the
expression of Ets-1, a known p16(INK4a) transcriptional activator, as
well as unknown Ink4a/Arf coregulatory molecules. These data suggest that
expression of the Ink4a/Arf tumor suppressor locus is a robust
biomarker, and possible effector, of mammalian aging.

Bull Cancer. 2004 May;91(5):399-402
[pRB, p53, p16INK4a, senescence and malignant transformation]
  Recent works aimed at clarifying the respective roles of p16INKa and
p14ARF (both located on the same INK4a locus on chromosome 9p21 in man)
in malignant transformation come to the conclusion that p16INK4a is the
true tumor suppressor gene in man. In mouse, it is the p19ARF knockout
that suppresses the barrier protecting cells from malignant
transformation. This situation is in agreement with p19ARF- and
p16-mediated senescence induced by oncogenic mutated ras (Ras*) in mouse
and man respectively. Other results have shown that senescence in human
diploid fibroblasts is associated with heterochromatin occurrence that
maintains in repressed state E2F1-induced gens required for G1 to S
phases transition. Since RB protein is responsible for this chromatin
modification, cells with any impaired RB pathway cannot enter into

Kidney Int. 2004 Feb;65(2):510-20
Expression of p16INK4a and other cell cycle regulator and senescence
associated genes in aging human kidney.
  BACKGROUND: Somatic cells in vitro have a finite life expectancy before
entering a state of senescence. If this state has an in vivo counterpart,
it could contribute to organ aging. We have previously shown that human
kidney cortex displays telomere shortening with age. In the present
study, we evaluated the relationship between renal age in humans and a
number of phenomena associated with cellular senescence in vitro.
METHODS: Human kidney specimens were obtained at 8 weeks to 88 years of
age and were assessed for changes related to aging. RESULTS: We found
that human kidneys expressed relatively constant levels of mRNAs for genes
potentially related to senescence. Among the candidate genes surveyed,
the cell cycle regulator p16INK4a emerged with the strongest association
with renal aging for both mRNA and protein expression. Proliferation as
measured by Ki-67 expression was inversely correlated with p16INK4a
expression, compatible with a role for p16INK4a as an irreversible cell
cycle inhibitor. Cyclooxygenase 1 and 2 (COX-1 and COX-2) mRNA expression
was elevated in older kidneys, associated with increased protein
expression. Comparison of gene expression with age-related histologic
changes revealed that glomerulosclerosis correlated with p16INK4a and
p53, whereas interstitial fibrosis and tubular atrophy were associated
with p16INK4a, p53, COX-1, transforming growth factor-beta 1 (TGF-beta
1), and heat shock protein A5 (HSPA5). CONCLUSION: We conclude
that some changes observed in cellular senescence in vitro do occur in
human kidney with age, particularly in the renal cortex, in some cases
correlating with histologic features. P16INK4a emerged with the most
consistent correlations with age and histologic changes and inversely
correlated with cell replication.

Circ Res. 2003 Oct 3;93(7):604-13. Epub 2003 Sep 04.
Senescence and death of primitive cells and myocytes lead to premature
cardiac aging and heart failure.
  Chronological myocardial aging is viewed as the inevitable effect of
time on the functional reserve of the heart. Cardiac failure in elderly
patients is commonly interpreted as an idiopathic or secondary myopathy
superimposed on the old heart independently from the aging process. Thus,
aged diseased hearts were studied to determine whether cell regeneration
was disproportionate to the accumulation of old dying cells, leading to
cardiac decompensation. Endomyocardial biopsies from 19 old patients with
a dilated myopathy were compared with specimens from 7 individuals of
similar age and normal ventricular function. Ten patients with idiopathic
dilated cardiomyopathy were also analyzed to detect differences with aged
diseased hearts. Senescent cells were identified by the expression of the
cell cycle inhibitor p16INK4a and cell death by hairpin 1 and 2.
Replication of primitive cells and myocytes was assessed by MCM5 labeling,
myocyte mitotic index, and telomerase function. Aged diseased hearts had
moderate hypertrophy and dilation, accumulation of p16INK4a positive
primitive cells and myocytes, and no structural damage. Cell death
markedly increased and occurred only in cells expressing p16INK4a that had
significant telomeric shortening. Cell multiplication, mitotic index and
telomerase increased but did not compensate for cell death or prevented
telomeric shortening. Idiopathic dilated cardiomyopathy had severe
hypertrophy and dilation, tissue injury, and minimal level
of p16INK4a labeling. In conclusion, telomere erosion, cellular
senescence, and death characterize aged diseased hearts and the
development of cardiac failure in humans.

Oncogene. 2000 Mar 23;19(13):1613-22
p16 INK4a can initiate an autonomous senescence program.
  The tumor suppressor p16INK4a is a potent mediator of cell cycle arrest
in transient expression studies, is induced in senescing cells, and can
impose morphological features of senescence. Nonetheless, it is unclear
whether p16INK4a can block cell proliferation irreversibly. We explored
this issue using osteogenic sarcoma cell clones with inducible p16INK4a
expression. Induction of p16INK4a for 1 day arrested most cells in G1
phase. If the induction was then interrupted, p16INK4a levels returned to
baseline and robust growth resumed within 3-5 days. When p16INK4a was
induced for 6 days DNA synthesis remained strongly inhibited and the cells
acquired morphological features of senescence. Moreover, if p16INK4a
induction was interrupted at this point and the cells were followed for
12 more days, most cells retained these morphologic features and either
failed to divide or died. This occurred despite the prompt return of
p16INK4a expression and retinoblastoma protein phosphorylation toward
baseline levels. In fact, some senescing cells appeared to enter S phase.
These results demonstrate that a sustained period of p16INK4a expression
is sufficient in this setting to impose a durable block to cell
proliferation and that this state becomes independent of p16INK4a
expression, hypophosphorylation of pRB, or a strict G1 arrest.

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