Week 12: Telomere Dynamics

Question 1

How is aging related to disease

Answer 1

1. aging is not merely a collection of diseases; With age we become more susceptible to certain diseases, but we also become more likely to die, frailer, and endure a number of physiological changes, not all of which lead to pathology.

Question 2

Normal cells are not immortal

Answer 2

1. 1950’s: Believed that cultivated cells could grow forever
   If not, then it was a result of a culturing deficiency
   In 1943, a cancer cell was grown in culture indefinitely
2. 1960’s: Leonard Hayflick noticed that human fibroblasts from embryonic tissue could only actively proliferate for several months. Theses cells ceased to grow but remained metabolically active

Question 3

Replicative senescence

Answer 3

1. Normal human fibroblasts have a finite replicative capacity in cell culture
2. Senescent cells are resistant to mitogenic stimulation (arrested in G1), Frozen cells thawed remembered their place in the PDL

Question 4

Mitotic clock

Answer 4

1. Phase I is the primary culture;
2. Phase II represents subcultivated cells during the period of exponential replication.
3. 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.

Question 5

Characteristics of senescent cells

Answer 5

1. Mitotically competent cells respond to various stressors (dysfunctional telomeres, non-telomeric DNA damage, excessive mitogenic signals; oncogenes) by undergoing cellular senescence.
2. The senescence response causes changes in cellular phenotype: permanent arrest of cell proliferation, development of resistance to apoptosis (in some cells), and an altered pattern of gene expression.

Question 6

Young vs senescent cells

Answer 6

1. Young:
   1a. Smaller cell volume with regular shape.
   1b. high cell density on plate
   1c. Some focal adhesions (green) attaching cell to substratum.
   1d. Some actin stress fibres (red).
2. Senescent:
   2a. Large cell volume fired egg appearance
   2b. low cell density on plate.
   2c. Many focal adhesions (green) attaching cell to substratum.
   2D. Extensive actin stress fibres (red).

Question 7

Loss of proliferating capacity w donor age

Answer 7

1. The PDL of dermal fibroblasts from various anatomical locations as a function of donor age.
2. Cells from newborns generally have a higher replicative capacity.
3. Keratinocyte stem cells loose their replicative capacity with increasing age.
4. Thinning of kertinocyte layer with increasing age.
5. Skin has less regenerative capacity

Question 8

Senescent cells occur in vivo

Answer 8

1. Aging is accompanied by the senescence of cells in the tissue.
2. Senescent cells in the benign precursors of melanomas (dysplastic Nevi).
3. Showing that cells are protected from neoplastic growth by the senescent program.
4. Thus, the escape from senescence is step in the development of all cancers

Question 9

Telomere loss of a model for replicative senescence

Answer 9

1. Human fibroblasts have a finite replicative capacity (Hayflick limit, 1961).
2. Telomeres shorten as normal human fibroblasts grow in culture.
3. Telomeres shorten as normal human fibroblasts grow in culture.
4. Telomeres shorten bc end replication problem

Question 10

Telomere structure

Answer 10

1. Telomeric DNA contains hundreds of tandem repeats of a hexa-nucleotide sequence.
2. One of the strands is G rich at the 3’ end, and it is slightly longer than the other strand (repeating G- rich sequence is TTAGGG: forms large duplex loops)
3. This looplike structure is formed and stabilized by specific telomere- binding proteins.
4. Such structures would nicely protect the end of the chromosome from degradation.
5. Telomeres are present at chromosome ends and consist of perfect repeats of TTAGGG over a length of 5-10 kilobase (kb) pairs in humans (Human telomeres have long G-rich 3’ single-strand overhangs that are heterogeneous in size (50-300 nt))

Question 11

The end replication problem

Answer 11

1. The free ends of linear DNA molecules introduce several complications.
   1a. First, the unprotected ends of the DNA at the end of a chromosome are likely to be more susceptible to digestion by exonucleases if they are left to freely dangle at the end of the chromosome during replication.
   1b. Second, the complete replication of DNA ends is difficult, because polymerases act only in the 5’ ->3’ direction….The lagging strand would have an incomplete 5’ end after the removal of the RNA primer….Each round of replication would further shorten the chromosome and slowly disappear

Question 12

What does the end replication problem cause

Answer 12

1. Shortening of telomeres
2. The primase enzyme is responsible for laying down short RNA primers…The gap can not be filled in by a conventional DNA polymerase….The gap will increase in length with each round of replication.

Question 13

Telomerase

Answer 13

1. Human telomerase is a reverse transcriptase that uses a RNA template to add telomeric repeats to the end of chromosomes.
   1a. In somatic cells, telomerase activity is low or absent.
   1b. In cancer cells and In stem cells, e.g. germ line, telomerase activity is high – maintains telomere length.
2. consists of 2 subunits (hTERT and hTR)
3. extends the 3’ end of telomeric DNA through nucleotide addition using hTR RNA as template; processive elongation

Question 14

Telomere length in tissues

Answer 14

1. At a critical telomere length a DNA damage response is invoked causing the cells to arrest, preventing further divisions (senescence).

Question 15

Shelterin complex

Answer 15

1. Human telomeres comprise three components: telomeric DNA, the shelterin complex and the telomerase complex.
2. Telomeric DNA has a single-stranded 3′ overhang: This overhang invades double-stranded telomeric repeats to form a t-loop structure that is crucial for telomere function.
3. Telomeric DNA protects chromosome ends through its association with the six-subunit shelterin complex.
4. The length of telomeric repeats can be maintained by telomerase, which is composed of TERT, TERC and several accessory proteins
   4a. TERT synthesizes telomeric DNA de novo using TERC as a template, whereas the accessory factors contribute to the biogenesis and nuclear trafficking of telomerase.

Question 16

Telomere erosion activates p53 through ATM & ATR

Answer 16

1. As cells approach replicative senescence, double-stranded telomeres shorten and the 3’ single-stranded overhang is eroded.
2. Telomere loop disruption (a consequence of telomere shortening, 3’ overhang erosion, loss of shelterin binding to telomeres) is the signal for senescence.
3. Dysfunctional telomeres activate a DNA damage response leading to p53 activation and replicative senescence.

Question 17

Crisis

Answer 17

1. There are 2 mortality barriers.
   1a. The first, M1 is senescence, the second is M2 Crisis.
2. Crisis occurs when senescence fails to initiate and telomeres continue to erode because the cell continues to undergo division.
3. The telomere sequence continues to shorten and eventually cannot support shelterin complexes at the terminus of the chromosomes.
4. The unprotected ends are sticky, and can fuse with other telomeric sequences leading to fusions.
5. These fusions lead to apoptosis as a result of genomic instability and loss of equal distribution of chromosomes during cell division.

Question 18

Extra

Question 19

Uncapping telomeres

Answer 19

1. Crisis can be artificially induced by introducing a dominant negative form of the shelterin complex protein TRF2.
2. This leads to the disruption of shelterin complex, leaving the chromosome ends unprotected.
3. In normal cells this is detected as a double stranded breaks which would lead to senescence and apoptosis.
4. In addition the unprotected ends can fuse with other telomeres leading to crisis.
5. The metaphase spread (B) demonstrates telomeric fusions as a consequence of TRF2 DN. The chromosomes become more life a bowl of noodles than pretzels.

Question 20

Shortened telomeres lead to dicentric chromosomes

Answer 20

1. In abnormal situations certain cells may acquire the ability to avoid senescence.
2. When cells bypass senescence and continue to divide, there will be variation of how quickly each telomere erodes.
3. Upon division some chromosomes will become unprotected (uncapped).
4. Unprotected chromosome ends are prone to fusion with other unprotected ends.
5. Since the short ends tend to be duplicated during S-phase and the same chromosome ends tend to be shortest, they can fuse together during mitosis.
6. This leads to a phenomena known as a dicentric chromosome during mitosis and an anaphase bridge at telophase.

Question 21

Shortened telomeres lead to breakage-fusion-bridge cycles

Answer 21

1. Fused chromosomes tend to break apart during chromatid separation.
2. These broken ends are than susceptible to fusing with other short telomeres or broken chromosomes because they are also unprotected.
3. These will again be torn apart in the next round mitosis
4. This process leads to translocations, deletions and duplications throughout the genome.
5. This is a frequent pathway during the development of cancer, since it can result in self selection of many characteristics of cancer including the stabilization of telomeres.

Question 22

Extra

Question 23

Telomere reactivation

Answer 23

1. Loss of the RB and p53 tumour suppressor pathways disables the ability of cells to respond with cell cycle arrest to ATR and ATM signalling.
2. As the cells continue to divide, their telomeres continue to shorten.
3. Once many telomeres become too short to function, the unprotected chromosome ends generate end-to-end fusions and dicentric chromosomes, leading to many forms of genome instability.
4. Ultimately, telomerase reactivation provides a route out of telomere crisis by healing critically shortened telomeres and improving genomic stability, thereby increasing cell viability.
   The resulting tumour will have active telomerase and a heavily rearranged genome.