Ch. 10: Eternal Life Flashcards
Most normal cells have a limited number of times
they can…
divide
Most human cells are a little more complex, but they
also have limited…
proliferative potential
If you take human cells out of the body and put them in a petri dish…
* They will…
continue to divide…
* And then they will stop dividing
senescence
when cells stop dividing and cannot divide again even when in an environment that favors growth.
* They exit the cell cycle irreversibly.
replicative senescence
is 1 type of senescence due to cells reaching their maximum
number of cell divisions.
Human cells will undergo stress-associated senescence mediated through …
the RB pathway
Cells with high levels of
physiological stress will increase levels of
p16
p16 will inhibit…
progression through G1 phase by inhibiting CyclinD-CDK4/6
(RB will never be phosphorylated and cells will never be able to go
past the R checkpoint.)
* Therefore…
cells with high p16 will
become senescent.
Human cells also undergo replicative senescence
mediated through…
the TP53 pathway
Cells with genomic damage
(including damage due to short telomeres) will increase levels of…
TP53
TP53 will inhibit…
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”).
What happens if human cells in culture lose TP53?
- Cells will undergo crisis:
- Widespread genomic
alterations/chaos. - Apoptosis is induced.
- Image on the right: Cell
population in crisis
refractile cells
catch the light and appear white- represent cell fragments in the middle of apoptosis.
How do human cells become immortal?
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.
What are telomeres?
- 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
Visualization of telomeres
- 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
telomeres actually look like (Ch. 10, slide 17)
- 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.
t-loop
Lasso-like structure at the end of a chromosome that
protects the ends of chromosomes from end-to-end fusion events.
Telomeres get ______ with every cell division
shorter
How do 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
end-replication problem
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
telomere biology
- 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
What happens if a telomere gets too short?
- 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.
Repeated breakage-fusion-breakage cycles will result in…
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.
Experimental evidence of end-to-end fusion events
- 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
Crisis is a result of repeated breakage-fusion-
breakage (BFB) cycles.
- If cells have short telomeres.
- And TP53 is not functional and cannot induce replicative
senescence. - Multiple BFB cycles will occur.
- Ultimately, cause apoptosis.
What is telomerase?
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
How do cancer cells activate telomerase?
- hTERT gene is actively transcribed into RNA and protein is made.
- Activation of the MYC (oncogene). A transcription factor that can activate hTERT gene expression.
- 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
- Mutations in promoter sequence of hTERT gene can be mutated as well, allowing for another transcription factor (ETS) to activate
Acquisition of immortalization in cancer cells
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
Human cells have a finite number of…
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.
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
Telomere biology is different in mice and humans
- 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).
What happens if you mate mTR-/- mice with each other?
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
Summary for mice
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.
Telomere restriction fragment (TRF) assay
- 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.
Telomerase Repeat Amplification Protocol
(TRAP) Assay
- 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.
