Bagian Biokimia Fakultas Kedokteran Universitas Hasanuddin Makassar

Dr. Marhaen Hardjo, M.Biomed, PhD

Biokimia Geriatri

Chromosome End Replication Problem

DNA replication and telomere shortening

The chromosome End ReplicationProblem

DNA polymerasesadd bases5’ -> 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)

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.

Telomeres

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 Can divide indefinitely.

Cells with a limited replicative lifespan. 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 limitHayflick limit: cells : cells are only capable of a are only capable of a limited number of limited number of population doublings in population doublings in culture.culture.

Here’s what is meant Here’s what is meant by the term doubling by the term doubling in in vitrovitro..

One serial passage or One serial passage or doubling of cellsdoubling of cells

Term is used to Term is used to describe replication describe replication going on in culture going on in culture dishes.dishes.

Organism + L.SOrganism + L.S: : Hayflick LimitHayflick Limit:-mouse about 3 years -doublings about 20-human about 100 -doublings about 40-60-Galapagos tortoise about 150 -doublings about 140

Cell proliferation potential greater in long-lived species

Population doublings

•Cells from older donors have “used up” some of doublings

Cell proliferation potential lower from older donors

Exponential SenescentSenescing

Senescence of keratinocytes

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-LÓM-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.

Telo

mer

e Le

ngth

(hum

ans)

Number of Doublings

20

10

Cellular (replicative) Senescence

Normal Somatic Cells

(Telomerase Negative)

Telomere also provide a means for "counting" cell division: telomeres

shorten with each cycleTelomeres shorten from 10-15 kb

(germ line) to 3-5 kb after 50-60 doublings(average lengths of TRFs)

Cellular senescence is triggered whencells acquire one or a few critically short telomeres.

• Telomerase mutants have a short lifespan.• When telomeres shorten to a critical point, yeast

cells stop dividing.

• Overexpression of telomerase:• Longer telomeres.• Increased replicative lifespan.

• Subtelomeric gene expression is supressed.• Shortening of telomeres relieves the supression.

Yeast replicative lifespan regulated by telomere length

• Lab strains of mice have very long telomeres.• 30-40kb telomeres.• Therefore, short telomeres aren’t 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.

Telomeres in mice

Dermal fibroblasts transformed with TERT (telomerase) continue dividing, Werner’s cells typically stop dividing at 20 population doublings.

Werner’s cellular phenotype reversed by telomerase

expression

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