0% found this document useful (0 votes)
19 views42 pages

Aging Lecture 5

Uploaded by

zhiqian343
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
0% found this document useful (0 votes)
19 views42 pages

Aging Lecture 5

Uploaded by

zhiqian343
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
You are on page 1/ 42

The Hayflick Limit, Telomeres and

DNA Instability
Fundamental Properties of Cells Lecture 5

Julian Pakay

"Senescence has no function--it is the subversion of function."


Alex Comfort
Two Concepts Important in Aging

1. Aging involves the accumulation of damage over time

2. There is a cellular basis for aging


Early Ideas on the Evolution of Aging

In 1889, he theorized that aging was part of life's


program because the old need to remove themselves
from the theatre to make room for the next
generation, sustaining the turnover that is necessary
for evolution.

But – this is a teleological explanation.

a purpose for aging has been identified, but not a


mechanism by which that purpose could be achieved.
August Weismann
Aging may have this advantage for the long-term
health of the community.

How would individuals acquire the genes that make


them get old and die?

Why would such individuals be more successful than


other individuals lacking such genes?
Aging is the Accumulation of Damage

Weismann dropped this concept and instead suggested that aging evolved
because organisms that segregate germ and soma must invest additional
resources to reproduce instead of maintaining the soma, and this
renunciation of the soma results in aging.
August Weismann – Programmed Cell Death

“death takes place because a worn-out tissue cannot forever renew itself, and
because a capacity for increase by means of cell division is not everlasting but
finite”

Main Idea

there is a specific limitation on the number of divisions that somatic cells


might undergo in the course of an individual life and that this number, like
the life span of individual generations of cells, is already determined in the
embryonic cell
Cell Division

Numerous checkpoints to
ensure DNA is accurately
duplicated

DNA replication occurs during S-phase


Controlling the Cell Cycle

Cyclins initiate DNA replication

• Extracellular mitotic signals


bind to the cell

• Result in nuclear mitotic signal

• Expression of cyclin/activation
of Cdk

• Phosphorylation of signalling
proteins to enter S phase
p53 helps prevent damaged DNA from being copied

• DNA repair mechanisms activate p53 kinase

• Phosphorylated p53 stimulates transcription of p21

• p21 binds to cyclin-Cdk complex and inactivates it


How many times can a cell divide?

“The cell is immortal. It is merely the fluid


in which it floats that degenerates. Renew
this fluid at regular intervals, give the cells
what they require for nutrition, and as far as
we know, the pulsation of life can go on
forever.”

Alexis Carrel, 1921


How long do cells live in culture?

Alexis Carrel (1921) suggested that all cells explanted in culture are
immortal, and that the lack of continuous cell replication was due to
ignorance on how best to cultivate the cells.

Claimed that chick heart fibroblasts grew continuously for 34 years.

Generally believed that all vertebrate cells could divide indefinitely in cell
culture.

The cells were fed with a daily extract of chick embryo tissue.

Do you believe this?

Cells fed with an extract of chick embryo tissue extracted under conditions that
permitted the addition of fresh living cells to the culture at each feeding.
Measuring life-span in culture

• Fibroblasts separated into individual cells by


trypsin digestion

• Cells placed in culture flask with medium

• Grown to confluence

• Again digested with trypsin and a few cells


are replated
Cultured cells can only
be passaged a limited
number of times

Though cancer cell


lines appeared
immortal – HeLa cells
Cultured Vertebrate Cells Are Not Immortal

Cultured normal human fibroblasts doubled a finite number of times,


after which the cells stopped dividing.

Hayflick mixed equal numbers of normal human male fibroblasts that


had divided many times (cells at the fortieth population doubling) with
female fibroblasts that had divided only a few times (cells at the tenth
population doubling). Unmixed cell populations were kept as controls.
Cultured Vertebrate Cells Are Not Immortal

When the male ‘control’ culture stopped dividing, the mixed culture was examined and
only female cells were found. This showed that the old cells ‘remembered’ they were
old, even when surrounded by young cells, and that technical errors or contaminating
viruses were unlikely explanations as to why only the male cell component had died
Life History of Mitotic Cells in Culture
Data From Fetal Fibroblasts For Several Species

• Varies with species


• Doubling rates do not correlate strongly with max lifespan
Hayflick Limit

• Phase I is the primary culture


• Phase II represents sub-cultivated cells during the period of exponential
replication.
• Phase III represents the period when cell replication ceases but
metabolism continues. Cells may remain in this state for at least one
year before death occurs
Phenotype of cells in Phase III (Senescent)
Phenotype of cells in Phase III
(Senescent)

Some begin apoptosis.


Some, though they no longer replicate,
remain metabolically active and generally
adopt phenotypes including flattened cell
morphology, altered gene expression and
secretion profiles (known as the
senescence-associated secretory
phenotype).

a | Young cells in phase II at population doubling 20.


b| Old cells in phase III at population doubling 55.
Not all Cells Show this
Replicative Senescence

• some mouse cells spontaneously


immortalize

• human and avian rarely spontaneously


immortalize but can be transformed by
transfection (viral)

• cancer cells

• cell lines
What causes this replicative senescence?

Hayflick’s laboratory showed that the replicometer was located in the nucleus
End Replication Problem

In the early 1970s it was realized that the properties of DNA


replication prevent the cells from fully copying the ends of linear
DNA.

Because of the nature of lagging-strand synthesis, DNA polymerase


cannot completely replicate the 3’ end of linear duplex DNA.

This was referred to as the end replication problem – James Watson


End Replication Problem

(A) Helicase separates the strand,


primase makes RNA primer required.

(B) DNA polymerase synthesizes DNA in


5’ to 3’ direction (leading strand).

(C) DNA polymerase moves backward on


lagging strand – makes DNA segments
– Okazaki fragments.
DNA polymerase, can not fully synthesize the 3'
end of linear DNA – needs a primer
The end-replication problem and senescence

Olovnikov, a Russian theoretical biologist, realized that this repeated


shortening of the DNA molecule at each round of DNA replication might
explain Hayflick’s finding that normal cells can replicate only a specific
number of times.
Telomeres – a protective cap

Telomeres prevent the ends of chromosomes


from fusing to each other and they allow
chromosome ends to attach to the nuclear
envelope in some species.

Sequence of hexameric repeats of TTAGGG


The telomeres in human cells also consist of
thousands of repeats
How Does Telomere Loss Lead To Senescence?

Why does this not happen all the time?


Telomeres – the protective cap
Telomere lengths shorten with cell division

Harley et al. (1990). Telomeres


shorten during ageing of human
fibroblasts.. Nature 345, 458-460

Telomere lengths shown not to be the same in all tissues.

How can some cells be immortal?


Telomerase

Telomerase – can repair


telomere attrition

Enzyme (reverse
transcriptase) with protein
+ RNA subunits

Adds telomeric repeats


directly to 3' overhang (uses
its own RNA component as
a template)

Vertebrate telomere repeat:


TTAGGG
Vertebrate telomerase RNA
template: AATCCC

Discovered by Carol Greider and Elizabeth Blackburn


Telomerase Expression

• Expressed by germ cells and early embryonic cells

• NOT expressed by most somatic cells (human)

• Expressed by certain stem cells, but highly regulated

• Expressed by 80-90% of tumour cells! (remaining 10-20%


still need to overcome end replication problem -- do so by
alternative telomere lengthening mechanism (ALT),
probably recombination )
Extension of cellular life-span by introduction of the catalytic subunit
of telomerase, hTERT. Fibroblasts were transfected with either an
empty vector or one containing the hTERT cDNA. Clones were
isolated and passaged continuously in cell culture.
Telomere Hypothesis of Aging

Telomeres shorten with age (T cells, tissue with high cell turnover)
Therefore, telomere shortening is a cause of aging

What is wrong with this hypothesis?

Telomere attrition contributes to aging ONLY IF replicative


senescence contributes to aging
Does Replicative Senescence Lead to Aging?
Could we gain immortality by using telomerase gene therapy?

Cells that express telomerase still undergo cellular senescence in response to


DNA damage, oncogenes, etc.

Cycloastragenol is a natural
ingredient extracted in minute
quantities from the root of the
astragalus plant. Its method of
action is to activate the hTERT
gene, thereby activating the
enzyme telomerase
Long-lived animals should have long telomeres
and
Short-lived animals should have short telomeres

Telomere length of a few different species:

Humans 5–15 kb
Mice Up to 150 kb
Rats 20–100 kb
Birds 5–20 kb
Ants 9–13 kb
Telomere length and maximum lifespan in mammalian species

Data from cultured


fibroblasts

If anything a negative correlation between telomere length and lifespan


Telomerase activity does not correlate with lifespan
Telomerase activity inversely correlates with body mass

Aging Cell. 2007 February ; 6(1): 45–52.


How can we explain these correlations?

Why might you want limit cell division in a large long-lived species?
How to explain these correlations?

One hypothesis:

Mice, for example, express telomerase in somatic and germline tissues,


while humans express telomerase almost exclusively in the germline. As
a result, when telomeres of human somatic cells reach a critical length
the cells enter irreversible growth arrest called replicative senescence.

Replicative senescence is believed to be an anticancer mechanism that


limits cell proliferation. Repression of telomerase in somatic cells has
evolved as a tumour-suppressor adaptation in large, long-lived
organisms.
What is the difference between aging and cancer?

cancer is the consequence of an aberrant gain of cellular fitness.

aging is the time-dependent accumulation of cellular damage


characterized by a loss of cellular fitness.

but

Cellular damage may occasionally provide aberrant advantages to


certain cells, which can eventually produce cancer. Therefore,
cancer and aging can be regarded as two different manifestations
of the same underlying process—namely, the accumulation of
cellular damage
Learning Objectives

• Understand the concept of replicative senescence (RS) and the Hayflick


limit.

• Explain the role of telomere attrition in RS.

• Understand the role of telomerase in extending the duration of


replication.

You might also like

pFad - Phonifier reborn

Pfad - The Proxy pFad of © 2024 Garber Painting. All rights reserved.

Note: This service is not intended for secure transactions such as banking, social media, email, or purchasing. Use at your own risk. We assume no liability whatsoever for broken pages.


Alternative Proxies:

Alternative Proxy

pFad Proxy

pFad v3 Proxy

pFad v4 Proxy