Ch. 10: Eternal Life

Question 1

Most normal cells have a limited number of times
they can…

Answer 1

divide

Question 2

Most human cells are a little more complex, but they
also have limited…

Answer 2

proliferative potential

Question 3

If you take human cells out of the body and put them in a petri dish…
* They will…

Answer 3

continue to divide…
* And then they will stop dividing

Question 4

senescence

Answer 4

when cells stop dividing and cannot divide again even when in an environment that favors growth.
* They exit the cell cycle irreversibly.

Question 5

replicative senescence

Answer 5

is 1 type of senescence due to cells reaching their maximum
number of cell divisions.

Question 6

Human cells will undergo stress-associated senescence mediated through …

Answer 6

the RB pathway

Question 7

Cells with high levels of
physiological stress will increase levels of

Answer 7

p16

Question 8

p16 will inhibit…

Answer 8

progression through G1 phase by inhibiting CyclinD-CDK4/6

Question 9

(RB will never be phosphorylated and cells will never be able to go
past the R checkpoint.)
* Therefore…

Answer 9

cells with high p16 will
become senescent.

Question 10

Human cells also undergo replicative senescence
mediated through…

Answer 10

the TP53 pathway

Question 11

Cells with genomic damage
(including damage due to short telomeres) will increase levels of…

Answer 11

TP53

Question 12

TP53 will inhibit…

Answer 12

progression through late G1 and early S by inhibiting active CyclinE-CDK2 and CyclinA-CDK2
- Therefore, cells with high TP53 will become senescent.
(“Replicative senescence”).

Question 13

What happens if human cells in culture lose TP53?

Answer 13

• Cells will undergo crisis:
• Widespread genomic
  alterations/chaos.
• Apoptosis is induced.
• Image on the right: Cell
  population in crisis

Question 14

refractile cells

Answer 14

catch the light and appear white- represent cell fragments in the middle of apoptosis.

Question 15

How do human cells become immortal?

Answer 15

1- inhibit RB pathway (allows them to avoid stress associated senescence)
* 2- could inhibit TP53 (allows them to avoid replicative senescence)
* 3- avoid genomic crisis/apoptosis (how do they do this?)
* Find a way to avoid genomic crisis due to short telomeres.

Question 16

What are telomeres?

Answer 16

• The physical ends of chromosomes.
• The DNA sequence is TTAGGG, repeated over and over again.
• Double stranded for 5-10 kilobases.
• Single stranded with a 3’ overhang for several hundred bases

Question 17

Visualization of telomeres

Answer 17

• If you do an in-situ hybridization.
• Make a fluorescent probe that is the reverse complement of the
  telomere sequence: AATCCC (it
  will base pair with TTAGGG) and you can visualize telomeres
• The ends of chromosomes

Question 18

telomeres actually look like (Ch. 10, slide 17)

Answer 18

• The 3’ “single-stranded” piece of DNA actually loops back and base- pairs upstream with the 5’ “double-stranded” piece of DNA
• Forms a t-loop: Lasso-like structure at the end of a chromosome that
  protects the ends of chromosomes from end-to-end fusion events.

Question 19

t-loop

Answer 19

Lasso-like structure at the end of a chromosome that
protects the ends of chromosomes from end-to-end fusion events.

Question 20

Telomeres get ______ with every cell division

Answer 20

shorter

Question 21

How do telomeres get shorter with every cell division?

Answer 21

Due to end-replication problem
* The inability of DNA replication machinery to copy the end of the 3’ strand
of DNA

Question 22

end-replication problem

Answer 22

The inability of DNA replication machinery to copy the end of the 3’ strand
of DNA

• DNA polymerase can only make a new DNA strand in the 5’ -> 3’ direction.
• DNA polymerase also needs an RNA primer to initiate new strand synthesis.
• Results in a lagging strand.
• When DNA replication machinery gets to the very end of the chromosome/DNA:
• The lagging strand primer is removed.
• The lagging strand primer was not placed at the exact 5’ end.
• Results in incomplete DNA replication of the 3’ parental strand

Question 23

telomere biology

Answer 23

• Telomeres get shorter with every cell division.
• Due to end-replication problem
• The inability of DNA replication machinery to copy the end of the 3’ strand of DNA
• Offers a molecular mechanism to “count” how many cell divisions
  a particular cell has undergone
• As telomeres get shorter know the cell has undergone more cell divisions

Question 24

What happens if a telomere gets too short?

Answer 24

• A t-loop wouldn’t be able to form
• The ends of our chromosomes would be linear
• Ligases, would fuse the ends of our chromosomes together
• = End to end fusion
• This results in a dicentric
  chromosome- 1 chromosome with 2 centromeres.

During cell division:
1 chromosome with no telomere region is duplicated.
The ends of the 2 sister chromatids will be ligated together.

Question 25

Repeated breakage-fusion-breakage cycles will result in...

Answer 25

genomic chaos * Can get fusion between 2 sister chromatids of the same chromosome. * Could also get fusion between 2 different chromosomes. * Would end up with daughter cells with extra copies of pieces of some chromosomes. * Would end up with daughter cells with loss of pieces of other chromosomes. * =genomic chaos (karyotype chaos) * Potential for loss of tumor suppressor genes. * Potential for gain of proto-oncogenes.

Question 26

Experimental evidence of end-to-end fusion events

Answer 26

* Cells without functional telomeres (unable to make t-loop) will result in chromosomes that undergo repeated end-to-end fusion events. Will eventually result in 1 huge chromosome. * 2 sister chromatids in anaphase, that are physically attached together. Eventually, they will physically be ripped apart at a weak point

Question 27

Crisis is a result of repeated breakage-fusion- breakage (BFB) cycles.

Answer 27

* If cells have short telomeres. * And TP53 is not functional and cannot induce replicative senescence. * Multiple BFB cycles will occur. * Ultimately, cause apoptosis.

Question 28

What is telomerase?

Answer 28

Main function: Telomerase can make telomeres longer! * Telomerase is a complex made of multiple proteins and an RNA. * The main protein with enzymatic activity is encoded by a gene called hTERT. * Telomerase uses the RNA as a template to add new DNA nucleotides to a single- stranded 3’ end of DNA. * Telomerase is a reverse transcriptase. Uses RNA as a template to make DNA

Question 29

How do cancer cells activate telomerase?

Answer 29

* hTERT gene is actively transcribed into RNA and protein is made. * 1. Activation of the MYC (oncogene). A transcription factor that can activate hTERT gene expression. * 2. Mutations in promoter sequence of hTERT gene can be mutated as well, allowing for another transcription factor (ETS) to activate transcription of the hTERT gene

Question 30

Acquisition of immortalization in cancer cells

Answer 30

In order for cancer cells to become immortal they must overcome: * 1- RB senescence pathway- induced by physiological stress * Inactivation of RB pathway 2- Replicative senescence- induced by short telomeres * Activation of telomerase

Question 31

Human cells have a finite number of...

Answer 31

cell divisions * They are mortal. * If you stick them in culture they will eventually stop dividing. * Due to senescence due to physiological stress (ex- ROS). * Due to replicative senescence due to short telomeres. * These mechanisms function to inhibit cancer formation.

Question 32

In order for cancer cells to become immortal they must overcome:

Answer 32

* 1- RB senescence pathway- induced by physiological stress * Inactivation of RB pathway 2- Replicative senescence- induced by short telomeres * Activation of telomerase

Question 33

Telomere biology is different in mice and humans

Answer 33

* Humans- Most cells do not express hTERT and do not have telomerase activity * Mice- Most cells express mTERT and have telomerase activity * As a result: * Mice have longer telomeres than humans. * If you culture mice cells in vitro they become immortal pretty easily (there is no replicative senescence barrier to immortalization).

Question 34

What happens if you mate mTR-/- mice with each other?

Answer 34

It would take 5 generations of breeding before you saw a difference in mouse phenotype. At the 5th generation without telomerase, their telomeres are short enough to cause aging phenotypes. Example- sickly, decreased capacity to heal wounds, widespread cell death, loss of tissue function

Question 35

Summary for mice

Answer 35

Mice are great models for lots of biological processes. Mice are great models for lots of processes related to human cancer. Mice are not great models for how human cells become immortally transformed.

Question 36

Telomere restriction fragment (TRF) assay

Answer 36

* Measures the lengths of telomeres in a cell. * Modified Southern Blot. * Chop up all the DNA in a cell using multiple restriction enzymes. * Telomere sequences do not have sites that restriction enzymes can recognize and chop up. * Have only telomeric DNA leftover. * Run this on a gel. Transfer to a sticky paper. * Use a fluorescent probe that base pairs with TTAGGG (AATCCC) to bind to telomeres to visualize the telomeres.

Question 37

Telomerase Repeat Amplification Protocol (TRAP) Assay

Answer 37

* Way to measure if there is active telomerase activity in your sample. * Make cell extract from your sample. * Add a fake telomere substrate to the cell extract. * If the cell extra has active telomerase activity it will add telomere sequences. * Perform a PCR to amplify the products.