Lecture 3: Telomeres, Cell Aging & Senescence

The “End Replication” Problem

Every time the cell divides (DNA replicates), the primers that were laid are not replaced.

Fragments of the DNA will get shorter overtime (20bp lost per cell division)

Can’t remake the entire chromosome on the lagging strand

Human Telomeres

3,000 repeats of the 5’ TTAGGG 3’ sequence

Telomerase

Has two parts:

1. reverse transcriptase protein (TERT)

2. rna template with the AAUCCC sequence (TERC); is species-specific

Telomerase in Action

RNA template AAU binds to DNA’s TTA (3’ of the lagging strand), then continues elongation of the telomere

Telomerase makes the 3’ longer than the 5’ end of the antiparallel strand = forms a T-loop (3’ end capping)

What does a T-loop (3’ end Capping) Typically Represent

A single-stranded DNA

ssDNA is a sign for other molecules to repair DNA

the T-loop is performed to hide the ssDNA and prevent hyper repair

Where are Telomeres Strongly Expressed/On?

In cells that maintain proliferation

stem cells

germ cells

proliferating cells (T cells, intestinal crypt)

cancer cells

Cell Division

Mitosis: the cell splits into two new daughter cells

Cyclins

promote progression through the cell cycle

the “license” the stages of the cell cycle

they partner with CDKs (cell division kinases)

Initiation of DNA Replication

1. Extracellular Mitotic Signal (i.e growth hormone) expresses S-cyclin

2. S-cyclin protein binds to CDK to activate it

3. A nuclear signaling protein (inactive) and phosphorous join the S-cyclin/CDK complex

4. All four molecules signal the nucleus to start replication

Protease degrade cyclin so that DNA replication occurs only once

G1 Checkpoint

Checkpoint for DNA Replication

G1 Checkpoint: p53 Pathway

p53 kinase senses DNA damage and sends a signal

DNA needs to be fixed before replicating it

1. p53 protein expressed p21

2. p21 inactivates the S-cyclin/CDK complex

p21 gets degraded by protease after DNA is repaired

p53

a tumor suppressor

induces cell-cycle arrest for damaged DNA

senescent cells use p53 to promote the arrested cell cycle state (arrest between G1 and S phase)

Hayflick Limit

cells have a limited ability to divide

Replicative Senescence

stops division

Cell Immortality is Possible in Culture

viral protein SV40T antigen integrating in the gene can lead to indefinite number of cell doublings = transformation

Must be transformed to grow forever

Tumorigenic

How to identify senescent cells

1. staining with beta-galactosidase (blue)

appear larger and flatter

decreased number of proteins associated with DNA replication, and overall decreased rate of protein synthesis

increase in pro-inflammatory cytokines

Mitotic Clock Theory

old cells sense short telomeres and induce cell cycle arrest

How do shortened telomeres cause somatic cell senescence?

short or uncapped telomere is read as a strand break

1. p53 is phosphorylated upon stress

2. activates p21

3. p21 inhibits the Cyclin E/CDK2 complex

4. Molecular brakes at G1 (cell cycle arrest)

This results in the cell becoming in a replicative senescence state

How do you start the cell cycle again if p21 made it go into arrest?

a mitogen signal from the outside starts the cell cycle back up again (hormones, steroids, etc)

Maintaining the Senescent State

p53 and pRb pathways are involved

these are tumor suppressor genes

pRb

retinolbastoma

works with p53 to maintain a senescent state for the cell due to telomere shortening

Cyclin/CDK complexes inhibit Rb

Inhibition of Rb (is hypo-phosphorylated) leads to cell cycle arrest

p16

like p53 where it inhibits Cyclin-CDK complexes, specifically Cyclin D-CDK 4,6

Triggers of Cellular Senescence (Telomere Shortening)

UV radiation

ROS (reactive oxygen species

nutrient imbalance

chromatin changes

oncogene activation

What does beta-gal. activity mean

senescence (blue color)

Telomerase Gene

hTRT

What did hTRT do experimentally

1. increased the doubling number (changed the Hayflick limit)

2. increased telomere length

Having long telomeres…

is associated with long life

Dyskeratosis congenita

a progeria (pro- premature + geras- old age)

defects in telomerase

high cancer

short lived (teens-50s)

Cancer & Telomeres

Active telomerase makes cells immortal, leading to a mass of cells (cancer)

What happens if you stop telomerase activity all together

disrupt fertility, wound healing, and the ability to fight infections

What does senescent cells lead to

1. decreased risk of tumorigenesis

2. increased inflammation (common feature of all age-related diseases)

Senescence-Associated Secretory Phenotype (SASP)

senescent cells result in a certain phenotype (SASP)

active senescent cells signaling (senescence messaging secretome, SMS) produce

1. cytokines

2. growth factors

3. proteases

Senolytics

class of drugs that selectively eliminate senescent cells

1. promote tumor growth

2. decrease inflammation (decrease risk of age-related diseases)