Biokimia Geriatri

Dr. Marhaen Hardjo, M.Biomed, PhD

Bagian Biokimia Fakultas Kedokteran

Universitas Hasanuddin Makassar

Chromosome End Replication

Problem

DNA replication and telomere shortening

The
chromosome
End
Replication
Problem
DNA
polymerases
add bases
5 -> 3 and
require a
primer
template

Consequences of the end

replication problem

One strand replicates to the end

The other strand has a 8 - 12 bp gap at the 5
end.
Each chromosome in a cell that divides
repeatedly will progressively shorten.
This will lead eventually to chromosomes
shorting until genes are lost from the ends.
Described by Olovnikow,1973.

Telomeres/telomerase maintain chromosome

ends

What are telomeres?

Telomeres are

Repetitive DNA sequences at the ends

of all human chromosomes
They contain thousands of repeats of
the six-nucleotide sequence, TTAGGG
In humans there are 46 chromosomes
and thus 92 telomeres (one at each
end)

What do telomeres do?

They protect the chromosomes.

They separate one chromosome
from another in the DNA sequence
Without telomeres, the ends of the
chromosomes would be "repaired",
leading to chromosome fusion and
massive genomic instability.

Telomere function, cont.

Telomeres are also thought to be

the "clock" that regulates how
many times an individual cell can
divide. Telomeric sequences
shorten each time the DNA
replicates.

Think of it like this.

Telomeres effectively "cap" the end

of a chromosome in a manner
similar to the way the plastic on
the ends of our shoelaces "caps"
and protects the shoelaces from
unraveling. (Geron corporation)

How are telomeres linked

to aging?

Once the telomere shrinks to a

certain level, the cell can no longer
divide. Its metabolism slows down,
it ages, and dies.

Chromosome Ends are specialized

structures called Telomeres

Blue = DNA

White = Telomere protein (TERT)

Telomeres

Repeated G rich sequence on one strand

in humans: (TTAGGG)n
Repeats can be several thousand basepairs long. In
humans, telomeric repeats average 5-15 kilobases.
Telomere specific proteins, eg. TRF1 & TRF2 bind to
the repeat sequence and protect the ends.

Telomere functions

Telomeres protect chromosome end

from DNA repair pathways, repair
leads to chromosomal fusions.

Maintain length of chromosomes.

Telomeres associate with the

nuclear membrane and maintain
nuclear organization.

Telomerase

Telomerase is a ribonucleoprotein enzyme

complex (a cellular reverse transcriptase).

TERT - RNA directed DNA polymerase.

TERC - RNA template.

It stabilizes telomere length by adding

hexameric (TTAGGG) repeats onto the
telomeric ends of the chromosomes, thus
compensating for the erosion of telomeres that
occurs in its absence.

Telomerase is composed of both RNA

and protein

How Does Telomerase Work?

Telomerase works by adding back

telomeric DNA to the ends of
chromosomes, thus compensating
for the loss of telomeres that
normally occurs as cells divide.
Most normal cells do not have this
enzyme and thus they lose
telomeres with each division.

The telomere theory of aging

Potentially immortal cells (germ cells,

cancer cells) maintain telomerase activity

Cells with a limited replicative lifespan.

Can divide indefinitely.

Should have no telomerase activity.
Progressively shortening telomeres.
Cell division serves as a mitotic clock for
replicative senescence.

Provides a mechanistic explanation for

the Hayflick limit.

Hayflick limit: cells

are only capable of a
limited number of
population doublings in
culture.
Heres what is meant
by the term doubling in
vitro.
Term is used to
describe replication
going on in culture
dishes.

One serial passage or

doubling of cells

Cell proliferation potential greater in

long-lived species
Organism + L.S:
-mouse about 3 years
-human about 100
-Galapagos tortoise about 150

Hayflick Limit:
Limit
-doublings about 20
-doublings about 40-60
-doublings about 140

Population doublings

Cell proliferation potential lower from

older donors

Cells from older donors have used up some of doublings

Senescence of keratinocytes

Exponential

Senescing

Senescent

Telomeres & Aging

Healthy human cells are mortal

because they can divide only a
finite number of times, growing
older each time they divide. Thus
cells in an elderly person are much
older than cells in an infant.

Telomeres & Aging

It has been proposed that telomere

shortening may be a molecular
clock mechanism that counts the
number of times a cell has divided
and when telomeres are short,
cellular senescence (growth arrest)
occurs.

Telomeres & Aging

It is believed that shortened

telomeres in mitotic (dividing) cells
may be responsible for some of the
changes we associate with normal
aging.

Think of it like this

Geron Corporation likens the

telomere and aging condition to this:

For the cell, having a long telomere can

be compared to having a full tank of
gas in your automobile; having a short
telomere is like running on empty. Each
time a cell divides, its telomeres
become a little shorter until the cells
simply can no longer divide (e.g., it
runs out of fuel).

Telomeres & Aging

After a certain number of cell

divisions, the telomeres would be so
short as to somehow prevent the cell
from further proliferation--putting it
in a state called senescence. In other
words, he proposed that telomere
length offered a clock for telling a
cell's longevity. - Scientific American

What next?

So, scientists have determined that

there is a direct connection
between telomere length and
aging. What was their next step?

What Next?

Dr. Jerry Shay and his colleagues (The

University of Texas Southwestern
Medical Center at Dallas ) found that
cellular aging can be bypassed or put
on hold by the introduction of the
catalytic component of telomerase
(i.e., the fuel added to the gas tank to
keep the car running)!

What is telomerase,
anyway?

Telomerase (TEE-LM-ER-ACE) is a
ribonucleoprotein enzyme complex (a
cellular reverse transcriptase) that has
been referred to as a cellular immortalizing
enzyme.
It stabilizes telomere length by adding
hexameric (TTAGGG) repeats onto the
telomeric ends of the chromosomes, thus
compensating for the erosion of telomeres
that occurs in its absence.

So how does this all link

together?

In the laboratory, cells in tissue culture

with introduced telomerase have
extended the length of their telomeres.
They have already divided for 250
generations past the time they
normally would stop dividing, and are
continuing to divide normally, giving
rise to normal cells with the normal
number of chromosomes.

How Does Telomerase

Work?

Telomerase works by adding back

telomeric DNA to the ends of
chromosomes, thus compensating
for the loss of telomeres that
normally occurs as cells divide.
Most normal cells do not have this
enzyme and thus they lose
telomeres with each division.

How Does Telomerase

Work?

In humans, telomerase is active in

germ cells, in vitro immortalized cells,
the vast majority of cancer cells and,
possibly, in some stem cells.
High telomerase activity exists in
germ cells, stem cells, epidermal skin
cells, follicular hair cells, and cancer
cells.

How Does Telomerase

Work?

Research also shows that the counter

that controls the wasting away of the
telomere can be "turned on" and
"turned off". The control button appears
to be an enzyme called telomerase
which can rejuvenate the telomere and
allow the cell to divide endlessly. Most
cells of the body contain telomerase
but it is in the "off" position so that the
cell is mortal and eventually dies.

How Does Telomerase

Work?

Some cells are immortal because

their telomerase is switched on
Examples of immortal cells: blood
cells and cancer cells
Cancer cells do not age because
they produce telomerase, which
keeps the telomere intact.

Visual Example

The next slide shows cells stained

to visualize the presence of
telomerase.
The bottom dish was treated to
produce active telomerase and is
still dividing
The top dish of normal cells of the
same age has stopped dividing

Telomerase and Cancer

There is experimental evidence

from hundreds of independent
laboratories that telomerase
activity is present in almost all
human tumors but not in tissues
adjacent to the tumors.

Telomerase and Cancer

Thus, clinical telomerase research

is currently focused on the
development of methods for the
accurate diagnosis of cancer and
on novel anti-telomerase cancer
therapeutics

Experimentation

Many experiments have shown

that there is a direct relationship
between telomeres and aging, and
that telomerase has the ability to
prolong life and cell division.

Genetic Link

The telomerase control gene has

been mapped to 3p21 (chromosome
3, the p (short) arm, locus 21)
Although the gene for telomerase is
present in all cells, hTRT is present
only in immortal cells, where it
serves to fuse the repeating
sequences of DNA to the
chromosomes, thereby lengthening
the telomeres.

Genetic Link

Proof that introduction of the hTRT

gene into mortal cells would cause
them to produce active telomerase
was offered in the December 1,
1997, issue of Nature Genetics by
Genron researchers

Telomerase Activity

In humans, telomerase is active in

germ cells, in vitro immortalized cells,
the vast majority of cancer cells and,
possibly, in some stem cells.
High telomerase activity exists in
germ cells, stem cells, epidermal skin
cells, follicular hair cells, and cancer
cells.
Inactive in most cells: somatic cells,
differentiated cells, post-mitotic cells.

Cellular senescence

Once the telomere shrinks to a certain

extent, the cell stops dividing.

~4kb in human cells triggers end to cell

division.

This leads to other changes called

cellular senescence:

Cell morphology changes.

Gene expression changes.

Telomere also provide a means for

"counting" cell division: telomeres
shorten with each cycle
Telomeres shorten from 10-15 kb
(germ line) to 3-5 kb after 50-60 doublings
(average lengths of TRFs)

Telomere Length (humans)

20

10

Cellular senescence is triggered when

cells acquire one or a few
critically short telomeres.

Normal
Somatic
Cells
(Telomerase
Negative)

Cellular (replicative) Senescence

Number of Doublings

Yeast replicative lifespan

regulated by telomere length

Telomerase mutants have a short lifespan.

Overexpression of telomerase:

When telomeres shorten to a critical point, yeast

cells stop dividing.

Longer telomeres.
Increased replicative lifespan.

Subtelomeric gene expression is supressed.

Shortening of telomeres relieves the supression.

Telomeres in mice

Lab strains of mice have very long telomeres.

30-40kb telomeres.
Therefore, short telomeres arent the cause of
senescence in mice!

Tert knock-out mice:

Normal for four generations as their telomeres

shorten,
Premature aging phenotypes present in the 5th
generation.

reversed by telomerase
expression

Dermal fibroblasts transformed with TERT

(telomerase) continue dividing, Werners cells
typically stop dividing at 20 population
doublings.

Telomerase and Cancer

There is experimental evidence

from hundreds of independent
laboratories that telomerase
activity is present in almost all
human tumors but not in tissues
adjacent to the tumors.

Telomerase and Cancer

Thus, clinical telomerase research

is currently focused on the
development of methods for the
accurate diagnosis of cancer and
on novel anti-telomerase cancer
therapeutics

Experimentation

Many experiments have shown

that there is a direct relationship
between telomeres and aging, and
that telomerase has the ability to
prolong life and cell division.

Genetic Link

The telomerase control gene has

been mapped to 3p21 (chromosome
3, the p (short) arm, locus 21)
Although the gene for telomerase is
present in all cells, hTRT is present
only in immortal cells, where it
serves to fuse the repeating
sequences of DNA to the
chromosomes, thereby lengthening
the telomeres.

Genetic Link

Proof that introduction of the hTRT

gene into mortal cells would cause
them to produce active telomerase
was offered in the December 1,
1997, issue of Nature Genetics by
Genron researchers