Cancer Biology Exam 2

What is the difference between gatekeepers vs. caretakers?

Gatekeeper: TSGs that affect proliferation, differentiation, and apoptosis. They play a role in allowing or disallowing cells to progress through the cell cycle.

• ex. APC, P21, PTEN, Rb, P53, etc.

Caretaker: DNA maintenance genes that affect cell biology indirectly by controlling the rate at which cells accumulate mutant genes.

What is cell fusion?

Taking a normal cell and cancer cell and creating a hybrid cell

• If the hybrid cell forms a tumor it is dominant (proto-oncogene)

• If the hybrid cell does not form a tumor it is recessive (TSG)

What is Rb?

Rb is a TSG that plays a role in the negative control of the cell cycle and in tumor progression.

• Those who are cured from Rb have a greater risk of contracting other cancers

Rb can be inactivated by mutation, insertion, and promoter methylation

What is the difference between sporadic and familiar forms of rb?

Sporadic: Unilateral and unfold (one eye infected)

Familiar: Bilateral and multifocal (both eyes infected)

• Those with this form have increased risk of other cancers bc the mutation is everywhere

Explain Knudson’s “two-hit” hypothesis

1. Familiar Rb (Germ-line mutation): 50% chance of inheriting mutation. A mutation in one copy of Rb is inherited in all body cells. (one parent passes down Rb and the child carries this mutation that is then inherited in all body cells)

• Cancer can occur early on in familial Rb.

1. Sporadic Rb (Somatic): No mutation is inherited but a mutation occurs in one of the body cells (Parent does not pass down the gene instead a mutation occurs)

What causes loss of Heterozygosity (LOH)?

1. Mitotic recombination: Recombination occurs and silences both copies

• two outcomes: (+/+) or (-/-)

1. Gene conversion: DNA Poly. copies the mutant cell

2. Chromosomal nondisjunction: Aneuploidy (non-normal # of chromosomes)

• One daughter cell retains both chromatids of chromosomes (triploid of chromosomes) - if there is an abnormal amount of chromatins the cell will delete one, typically the normal chromatid which causes cancer.

What is esterase D in relation to Rb?

Esterase D is used as a marker for Rb (Tells us whether or not Rb has been deleted) since it is located close to Rb. If Rb gene locus is lost due to LOH, then esterase D locus should also be lost

What is RFLP? What does EcoRI have to do with this?

This is a technique where single base or insertion/deletion polymorphisms lie within restriction enzyme recognition sites. This can be used to identify localization of TSGs through the detection of variations in the lengths of restriction fragments.

EcoRI acts as molecular scissors and cuts specific DNA sequences. It cuts up DNA into fragments of varying lengths

What is the difference between genetic and epigenetic alterations?

1. Genetic: Mutations —> LOH

a. Mitotic recombination

b. Gene conversion

c. Chromosomal nondisjunction

1. Epigenetic modification: Non-genetic changes that cause inactivation of TSG

a. hypomethylation: TSGs cannot bind (loss of function)

b. Hypomethylation: Proto-oncogenes are always active (gain in function)

More than half the TSGs are found to be silenced in SPORADIC cancers by promoter methylation

What is DNA Methyltransferase (DNMT) enzyme?

DNMT is responsible for causing hypermethylation during tumor development and can be detected with specific antibodies. The level of DNMT increased with increased tumor progression.

What is In Situ Hybridization (ISH)?

This is a technique used to measure DNA methylation (how much DNA methylation is occurring). It uses a methylation-specific probe for p16INK4A(TSG) promoter.

What is the cell cycle clock?

The cell cycle clock is defined by growth and division. External signals influence a cells decisions to enter into the active cell cycle.

There are specific checkpoints where the cell cycle stops until a go-ahead signal is received. Once cells decide to divide it is a ONE way direction, so they can only kill or repair once started.

What is the difference between the G1 checkpoint vs. G0 checkpoint?

G1: This starts the cell cycle and if the cell receives the go-ahead here then the cycle continues

G0: This is known as cell cycle arrest. This happens if a daughter cell does not receive the go-ahead at the G1 checkpoint and must arrest entering a non-dividing state.

• cells enter G0 in the presence of anti-mitogenic factors or the absence of mitogenic GFs

What determines how cells go to different checkpoints in the cell?

This is typically determined by TSGs. The G1 checkpoint is the MOST important because it checks if DNA is okay and is the ONLY checkpoint dependent on growth factors.

How does Akt inhibit TSC complex protein?

A change in PIP2 to PIP3 activates Akt. Akt blocks TSC1 (TSG) and this blockage allows for activation of Rheb, which allows for more MTOR, which activates PS6 kinase.

Akt blocks autophage (protects against Neuro diseases) by activating MTOR.

What is the Restriction point (R Point)?

This is a period during the G1 checkpoint where the cell must decide to continue through the cell cycle, remain in G1, or go into G0.

This is determined by mitogenic GFs and TGF-B

What are the different cell cycle checkpoints? What do they check for?

G1: Checks for DNA damage

S: Checks for DNA damage

G2: Checks for DNA damage and replication

M (Mitosis or Metaphase): Checks for proper chromosome alignment

What are the consequences of losing checkpoint controls?

1. If Bud1 checkpoint protein is lost then chromosomes that are not ready to be separated.

   1. Bud1 prevents separation of chromosomes that are not ready to separate.

2. Is Rad 17 checkpoint protein is lost, Rad1 prevents chromosomal re-replication. This can increase genomic instability (increasing the ploidy of the cell)

3. If ATR and Rad3 checkpoint protein kinase are absent this leads to fragile sites of chromosome that are prone to breakage. - typically DNA replication is halted until replication fork is repaired.

• Fragile sites on human chromosomes 3 and 16 are apparent when lacking ATR

What is one of the things that make the cell cycle work?

Cyclin-dependent kinases (serine/threonine): CDKs work as catalytic subunit and are dependent on the association with the regulatory subunit cyclin for activity. (CDKs and Cyclins form a complex)

1. Upon association, cyclins serves as guide dogs for (D1, D2, D3) CDKs to recognize appropriate substrates.

How did scientist find out about the relationship between cyclins and the cell cycle?

Scientist analyzed cell cycle-dependent fluctuations in cyclin B levels.

• They found Cyclin B fluctuated in embryos (high peaks in the s phase & drop in mitosis). Fluctuations were noticeable because in early embryos the cell cycle is synchronous aka they enter the M phase at the same time

How are cell cycle fluctuations well-programmed?

The collapse of various cyclin species as cells advance from one cell cycle phase to the next is due to their rapid degradation, being triggered by the action of highly coordinated ubiquitin ligases.

Cyclin D helps DNA pass the R point and is controlled by extracellular signals (Growth factors).

• Cyclin D increases with extracellular signals

What are the different Cyclin Ds? Why are there different types?

There is Cyclin D1, D2, and D3. Mammalian cells express cyclins based on tissue-specific patterns. They are under the control of transcription factors (TFs) to provide fine tuning of regulation.

• It enables diverse sets of extracellular signals to influence the activities of CDK4/6

What are the two types of CDK Inhibitors?

1. INK4 (active in early and mid-G1) - Activates p15 and p16 which bind to the cyclin D- CDK4/6 complex. This causes the cyclin binding site to be disregulated and the affinity for cyclin D is reduced.

1. Cip/Kip (active all throughout the cell cycle) - Activates P21, p27, and p57 which bind to Cyclin E- CDK2. This causes the ATP binding site in the catalytic cleft of CDK2 to be obstructed.

How are CDK Inhibitors regulated?

TGF-B regulates the expression of CDK Inhibitors. TGF-B is a growth factor can activate p15(strong) or p21(weak).

• Activation of p15: TGF-B can inhibit cells that have not passed the R point

• Activation of P21: TGF-B can inhibit cells that have passed the R point (metabolic stress, DNA damage, or hypoxia)

How do mitogens control the cell cycle?

Mitogens control cell cycle machinery through activation of Akt/PKB, causing the phosphorylation and cytoplasmic localization of p21^Cip1 and p27^Kip1.

• Akt phosphorylates p21 and p27 so they don’t translocate to the nucleus (promotes cell survival because CDK Inhibitors are phosphorylated)

PTEN is a phosphotase that removes phosphates from PIP3 (more p21 and p17 into the nucleus with this)

What is dominant-negative?

Dominant-negative is a mutation that causes a protein to lose function due to the “poisoning” of subunits.

• ex. PIP3 can bind but Akt is turned off because there is no kinase binding site (T157)

What are the actions of p21 and p27 in R point transition?

Early G1: High levels of p21 and p27

Early/mid and mid/late G1: D-CDK4/6 complexes accumulate due to mitogens (p21 and p27 are attaching to complexes) but this binding does not inhibit p21 or p27. Sequester p21 and p27 form E-CDK

Late G1: E-CDK2 is liberated and is allowed to trigger the R point transition and phosphorylation events during G1-S phase.

How do the cells go from G1 to S phase? What governs the R-point Transition?

pRb is the molecular governor of R-point Transition. The phosphorylation of pRb decides the movement to the next part of the cycle.

pRb conformation is changed by CDKs adding phosphates, which then bind to its own subset of E2F transcription factors.

Describe the difference between hyperphosphorylated and hypophosphorylated Rb.

Hyper: This is the phosphorylation of Rb and results in the cell moving past the R point

• Presence of oncoproteins can mimic this by preventing pRb from binding to E2Fs.

Hypo: This is typically seen being binded to oncoproteins (Ad5, E1A, HPV E7, SV40) and is an inhibitor of the cell cycle.

What are E2Fs? Describe the family of E2Fs.

E2Fs are associated with the promoters of a number of genes. There are 8 members in the E2Fs family.

Repressor E2Fs are activated by E2Fs 1, 2, and 3 to inhibit their actions (negative feedback loop)

E2Fs 4 and 5 are involved in repression in early/mid-G1

E2Fs 7 and 8 are involved in repression in late G1 at the G1/S transition

How does the connection between E2F/DP and Rb control the cell cycle?

In G1, in the presence of low level of CDKIs, active CDK/cyclin complexes trigger the hyperphosphorylation of Rb, releasing the dimeric transcription factor E2F/DP and thereby inducing the transactivation of genes with functional E2F-binding sites (for example, growth and cell cycle regulators and genes encoding proteins required for nucleotide and DNA biosynthesis).

• E2Fs 1, 2, & 3 attract HAC (Histone acetylases) to remodel chromatin and recruit RNA polymerase to initiate transcription.

How does the binding of hypophosphorylated pRb to E2Fs affect the cell cycle?

Hypophosphorylated pRb binds to E2Fs and blocks the transactivation domain of E2Fs.

It then recruits HDAC (Histone deacetylase) to the promoter by pRb to actively repress transcription.

In quiescent G0 cells expression of E2Fs 4&5 are present in abundance. Yet, E2Fs 1, 2, & 3 are more present in proliferating cells

Describe the feed forward loop and how it ensures the irreversibility of cell cycle advance

1. Cyclin E adds more phosphates the pRb. With more E2F, more Cyclin E is created allowing for this forward loop

2. E2F can activate Skp2 which marks proteins for degradation (p27)

3. Cyclin E binds to p27 which leading to p27 degradation, which removes the “brake” on cell cycle arrest

Describe the pathway for cell cycle control using Cyclin D.

1. Growth factors bind to growth factor receptors. This activates Ras, which then activates Cyclin D and E. The presence of Cyclin D and E cause pRb to be inactivated, which activates E2Fs and allows the cell to move into S phase.

2. Mitogens activates Ras which can result in:

   1. If Ap-1 and PI3K are activated this stimulates cyclin D

   2. If GSK-3b or Erk are activated this inhibits cyclin D

What is the role of myc?

Myc is a helix +loop + helix transcription factor. Myc level is strongly influenced by mitogenic signals

Cells that overexpress Myc tend to be in a state similar to cells lacking pRb function, which shows that deregulation of passage through R-point can occur by Myc.

What is the role of Myc in proliferation and differentiation?

Proliferation: Myc must bind with Max

Differentiation: To shut the system off and increase differentiation, Mxd blocks Myc and binds with Max

How does Myc effect the cell cycle clock?

1. Myc-Max: Functions as a transcription activator.

   1. Activate D2 and CDK4, leading to hypophosphorylation of pRb and p27 liberating E-CDK2

   2. Activate ubiquitin ligase (Skp2) promoting ubiquitination and degradation of p27

   3. Activate E2F1, 2, and 3

2. Myc-Miz1: Functions as a transcription repressor

   1. Repress the expression of p15 and p21 that confers resistance to TGF-B

How does TGF- B ensure its growth inhibitory action in normal cells?

1. Smad3/4-miz1 form a complex that activates p15 and Rb will not be phosphorylated

2. Smad3 can bind to E2F4/5 and repress Myc. Myc is prevented from repressing the expression of those two CDK Inhibitors through the formation of the Smad3-E2F4/5-p107 complex.

How do cancer cells evade TGF-B imposed growth inhibition?

1. Overexpression of Myc - Proliferation doesn’t stop

2. Mutations in the Myc promoter: Myc is constitively expressed, and no longer responsive to TGF-B induced repression

3. Smad2 mutation or mutation in TGFB-RII (not as important)

Provide an examples for how blocked differentiation can accompy tumor progression.

Id2 are transcription factors activated by Myc to inhibit differentiation of cells.

• Id2 binds with MyoD and blocks differentiation

Id proteins lack DNA binding domain and act as natural “dominant negative” Inhibitors of bHLH proteins.

How can cyclin E impact cancer progression, specifically breast cancer?

High levels of cyclin E drives deregulation of pRB phosphorylation and inactivation

High levels of cyclin E are also associated with abnormal centrosomes that results in genomic instability and acceleration of tumor progression.

• Chromosomal breaking or aneuploidy can occur

• Normal cells have either one when not dividing or two when dividing

How do tumor cells benefit from the P53 mutant allele rather than null alleles?

Mutant p53 enhances tumorgenicity in the absence of the transdominant negative mech.

Mutant p53 can also regulate the expression of endogenous c-myc gene and is a potent activator of the c-myc promoter.

This plays a role in the gain-of-function phenotype

How can mutant p53 proteins foster tumor cell formation?

Mutant p53 collaborates with ras to induce transformation

Name some facts about p53

1. p53 contains 5 domains. In addition to MDM2 binding transactivation domain and DNA binding domain, it also contains an oligomerization domain (allows it to form tetramers) at the C-terminus.

2. Most of p53 mutations are located within the DNA binding domain.

3. P53 works as a transcription factor which binds as a tetramer to the p53 consensus sites.

How does p53 function as a dominant negative allele?

p53 mutant can act as a DN having one wild-type and mutant still complex and not functional. This would eliminate one-half p53 function (wt allele will compensate by making more of the wt protein).

Mutant embryonic stem cells show that the cells with mutant allele present in heterozygous configuration showed substantial reductions in p53 function, as compared to the ES cells lacking one of the two p53 gene copies.

What can cause p53 signaling to activate?

A variety of cell-physiologic stresses can cause a rapid increase in p53 levels. DNA damage and de-regulated growth signals cause p53 stability.

The accumulated p53 protein induces a number of responses through transactivation of downstream target genes. Among them, the cytostatic and pro-apoptotic powers of p53 represent a major threat to incipient cancer cells.

What is MDM2?

MDM2 (oncogene) regulates p53 but it is also activated by p53. They have a negative feedback loop

MDM2, a downstream target gene of p53, works as an E3 ligase which binds p53 and facilitates ubiquitin-mediated degradation of p53.

MDM2 antagonizes p53 and prevents entrance of a cell into cell cycle arrest or into apoptotic suicide program.

What are the kinases that control p53 levels?

Chk2, ATM, and ATR - These kinases prevent the interaction between p53 and MDM2

ATM and ATR can directly phosphorylate p53 but can also indirectly through activation of Chk1/2

What is the relationship between MDM2 and p53?

In normal cells, MDM2 keeps p53 levels very low

Positive: Cell survival signals can trigger Akt/PKB that can phosphorylate mdm2 and facilitate its nuclear translocation.

Negative (normal): Under stress, DNA damage-sensing kinases (ATR & ATM) phosphorylates p53, and prevents the binding of MDM2 to p53. They can also phosphorylate and inactivate MDM2 preventing the binding of MDM2 to p53.

What is the role of ARF?

ARF can also block MDM2 but only functions when p53 is present.

ARF binds to MDM2, which is created by p53, and isolates MDM2 in the nucleolus, which inhibits its action and causes p53 to be expressed at an increased level.

How can E2F affect ARF? How can ARF control apoptosis?

Induced E2F activity can lead to increased ARF expression.

Some oncogenic signals favor apoptosis through their ability to induce E2F activity, which leads to increased ARF expression, suggesting the involvement of this signaling in the elimination of cells with overly active E2F signaling.

How can E2F induced apoptosis be used for cancer?

E2F-induced apoptosis may serve as an anti-cancer mechanism to eliminate unwanted, pre-neoplastic cells.

What is the difference between apoptosis and necrosis?

Apoptosis (cell programmed death): Initiated by the activation of a suicide program within cells that lead to fragmentation of chromosomes, shrinkage of cytoplasm and death without lysis or damage to neighboring cells.

Necrosis: Type of death due to pathological process (injury related), in which cells break up releasing contents into surrounding environment.

What are the hallmarks for apoptosis? What are ways that we can see if cells are going through apoptosis?

1. Cell fusion

2. Chromatin condensation (condensed small nucleus)

3. Formation of DNA ladders

4. Fragmented golgi bodies

Staining that shows phospholipids, A TUNEL Assay (chromosomal DNA becomes fragmented), or immunostaining (that shows an increase in caspases)

What are some triggers for apoptosis?

1. Death triggers

2. Physiological triggers: Growth factor deprivation, TNFa, FasL, and loss of attachment

3. Damage related signals: radiation, anticancer agents, oxidants, bacterial toxins

What are the biological functions of apoptosis?

1. Sculpting the embryo

2. Maintaining the tissue homeostasis

3. Terminating immune responses

4. Restricting the progress of infections

5. Eliminating damaged cells

Describe the IgG promoter with myc vs. Bcl. Describe the technique done with mice using these two

IgG-Myc is key for cell proliferation - Myc will remain turned on

IgG-BcL is key for pro-survival

Cloned genes of IgG-myc and IgG-Bcl were injected into fertilized mouse eggs. These eggs were transplanted into females. Bred IgG-Myc mouse and IgG-Bcl mouse which forms three groups

• IgG-Myc only: Only die faster from cancer (continued proliferation)

• IgG-Myc + IgG-Bcl: Die faster

• IgG-Bcl only: Survive longer (pro-survival)

What is the role of the mitochondria?

The mitochondria is important for killing the cell. It must constantly be recycled (mitophagy)

1. Reactive Oxygen species (ROS): This is not controlled because it relies on food

2. Apoptosis: Cytochrome C resides in the inner and outer mitochondrial membranes. When signals trigger the initiation of apoptosis, the outer mitochondrial membrane becomes depolarized and Cytochrome C is released into cytosol.

What is the role of Cytochrome C?

Cytochrome C releasal from the membrane into the cytosol singals for apoptosis. When it is released it associated with other proteins to trigger a cascade of events yielding apoptotic death.

What are the pro and anti-apoptotic Bcl-2 family proteins? What is the relationship between these proteins?

Bcl - anti-apoptosis (pro-survival)

Bax and Bak - pro-apoptosis

Both pro and anti-apoptotic genes share a common BH3 domain. The balance between the two is important (dose dependent)

Anti-apoptosis (Bcl-2 family) proteins block Bax & Bak to stop apoptosis but pro-apoptosis genes can also block anti-apoptosis proteins so apoptosis can occur.

Describe how pro-apoptosis genes work in the mitochondria

If apoptosis is initiated, Bak merges with Bax in punctuate foci on the surface of the mitochondria, which participated in mitochondrial fragmentation and contributes to Cytochrome C releasal.

How is apoptosis controlled via anti-apoptotic gene (Bcl-2)? How is apoptosis happening via pro-apoptotic genes?

Bcl keeps Cytochrome C inside the mitochondria. Bcl-2 must be present in sufficient concentrations to sequester pro-apoptotic BH3-only proteins (Bim, Bad, Bax, Bak) and inhibit apoptosis.

Yet, if pro-apoptotic BH3-only proteins are in relative abundance, they stimulate the homo-oligomerization of Bax and Bak. This association promotes mitochondrial outer membrane permeabilization which triggers apoptosis.

How does Akt promote cell survival?

When Akt is activated it phosphorylates Bad (BH3-only proteins) and Bad cannot bind to Bcl-2, which allows Bcl-2 to bind with Bax and inhibit apoptosis.

How is the activation of pro-apoptotic BH3-only proteins controlled?

1. Bim is regulated at post-translational level via phosphorylation by Erk, which results in its ubiquitylation and degradation.

2. Bad is phosphorylted by anti-apoptosis kinase (Akt/PKB or Raf) which leads its sequestration by 14-3-3 protein and loses its ability to bind the anti-apoptotic Bcl2 protein

3. Bid protein is liberated from inhibitory domain by action of protease that activates caspace and granzymes released.

What is the role of caspases in apoptosis?

*Caspase 3 and 9*

Caspases control cell death - they are a family of cysteine proteases that cleave protein substrates after specific aspartate residues

What is the role of Apaf1 in apoptosis?

Apaf1 (apoptotic protease activating factor 1). Bcl can inhibit Apaf1 which triggers caspase 9 and 3 to trigger apoptosis

What are the two intrinsic pathways for apoptosis?

1. Apoptotic Caspase cascade: When Cytochrome c is released Apaf-1 becomes organized and its role is to recruit caspase-9, which cleaves caspase-3 and triggers cell death

2. SMAC/DIABLO can also be released with Cytochrome C. With its releasal it inhibits IAPs (Inhibitors of apoptosis) which triggers caspase 3 and triggers cell death

What are the extrinsic pathways for apoptosis?

Death receptor and Ligand : Receptor triggers Fas-associated death domain (FADD) which cleaves caspase 8 and triggers the caspase cascade that triggers apoptosis

1. FADD forms a complex with DISC (death-inducing signaling complex)

2. Cancer cells (with Fas receptor) are attacked by cytotoxic T lymphocytes (with FasL)

What is the difference between intrinsic and extrinsic apoptotic pathways? How does the conversion of these pathways take place.

Intrinsic (stress-activated or mitochondrial pathway): Triggered by DNA damage, Hypoxia, imbalance in signaling pathways or excessive oxidants.

Extrinsic: Important for immune homeostasis and function, death signal originates from outside the cell, receptor-mediated activation, Ligands include (FasL/CD95L, TNF-a, APO2L/TRAIL)

Extracellular signal can trigger intracellular signal —> Executionar caspases 3, 6, 7 can also send signals to the mitochondria to trigger Cytochrome C release (through the activation of BH3-only proteins), which then triggers the apoptotic caspase cascade.

How is apoptosis activated by p53?

1. P53 can activate Fas (cell death ligand)

2. P53 can activate Bax (this triggers Cytochrome C releasal)

3. P53 can activate Insulin growth factor binding protein (IGFBP-3)

   1. This binds with insulin growth factor and blocks its binding with insulin growth factor receptor which inhibits Akt

Either transcriptional activation of pro-apoptotic genes or transcriptional repression of anti-apoptotic genes.

What is autophagy? What is its role in cancer?

Autophagy (second death program): Plays a key role in the elimination of undesired cells in various tissue throughout the body. It is often activated when cells suffer nutrient starvation and digest their own intracellular organelles in cytoplasmic lysosomes.

Can also be used as a means of eliminating cancer cells. Beclin-1, a key autophagy-promoting protein.

Why do normal cells have limited number of population doubling?

Cells exhibited limited number of replicative cycles and then entered into a state known as senescence.

What is senescence? What are the characteristics of a cell undergoing senescence?

Senescence is a way to prevent cancer and this is when cells stop dividing but remain metabolically active.

• Metabolically active, but cannot re-enter into active cell cycle

• Spread out in monolayer culture

• Acquire large cytoplasm

• Persists as long as they are given growth factors

• Display cell surface growth factor receptors but downstream signaling pathways are inactivated by unknown mechanisms

• Express senescence-associated acidic B-galactosidase enzyme

What factors affect the number of replicative doublings or population doubling?

1. Species from which cells were prepared

2. The tissue of origin

3. Age of the donor organism

Why do normal cells lack immortalized growth properties?

Early in embryogenesis, cells have unlimited replicative potential, but during differentiation specific cell lineages have limited post-embryonic population doublings.

Immortality of cancer cells is due to telomeres! In normal cells telomeres shorten after each division and when they become too short the cell cannot divide further (senescense)

What is the difference between Senescense and crisis?

1. Senescense: Cumulative physiologic stress over extended periods of time halts further proliferation.

   1. Accumulation of oxidative damage

2. Crisis: Cells have used up the allowed “quota” of replicative doublings. When they enter this state it leads to apoptosis.

   1. Occurs if senescense isn’t enough

Describe how they know senescence happens using phsyologic stresses.

1. They had two plates: One with feeder cells (fibroblast) and another without and they analyzed p16 to see the dependence of epithelial cells on fibroblast to survive and proliferate.

• With plates: stopped dividing after a while

• With feeder: Continues to divide (fibroblast are doing something that cells the cells to keep dividing).

2. Species in 3% O2 (living conditions) have continued dividing while those in 20% O2 stop dividing after a while

3. No p53 or P16 when nonstop dividing is seen - P16 over expression looks similar to senescence.

What genes influence the culture conditions on the onset of senescence?

1. P15 and P16 mainly block G1

   1. Cyclin-CDK4 is inhibited and Rb is not phosphorylated and the cell can enter senescence.

2. P21 blocks G1

   1. Inhibits cyclin E-CDK2 which prevents Rb phosphorylation.

What is the role of p53 and Rb in senescence? What technique shows this.

Both p53 and Rb inactivation are required to avoid senescence.

• With both senescence will occur.

Using SV40-LT, to block both p53 and Rb. They transfected three cells

1. HEK cells + no p53:

2. HEK cells + no Rb:

3. HEK cells + no p53, no Rb: ends up bypassing senescence and has continued growth.

How do they know senescence actually occurs? Why does a mutation in TSG lead to senescence (Use BRCA1 as an example)?

In-vivo testing shows that senescence actually occurs — staining with SA-beta-gal shows an increase in blue which shows senscence

BRCA1 is key for DNA damage and without BRCA1 there will be an influx of DNA damage which leads to senescence.

How do cells pass crisis?

1. Loss of telomeres causes crisis

   1. Telomeric DNA: This is a single extremely long DNA molecule and this prevents end-to-end fusion of chromosomal DNA

   2. DNA transfection: linear DNA gets end-to-end fusion by nucleases and DNA ligases

What is the role of telemeric DNA in normal vs. cancer cells?

In normal cells, the shortening of telomeric DNA is in concert with cell proliferation that will then trigger cell death when too short.

• telomeric shortening is the measure of cell generations as they pass down from the the early embryos

In cancer cells, telomeric DNA length is maintained

• Recovery from CRISIS happens due to maintenance of Telomeric DNA length

Why does the shortening of telomeric DNA lead to genomic instability and apoptosis?

Telomere erosion causes Breakage-Fusion-Bridge (BFB) cycle and chromosomal translocation.

• This can cause truncation, translocation, and aneuploidy

BFB cycle - Unprotected chromatid ends that allow for end-to-end fusion and when anaphase occurs the pulling of these chromatids causes weird breaking

Telomere shortening results from end-replication problem in telomeric DNA. The lagging strand causes telomeres to get shorter after each replication.

What is telomerase? What are two telomerase holoenzymes and their roles?

Telomerase is an enzyme that maintains telomeres length (elongates telomeric DNA), which is key for stopping crisis

1. Human telomerase reverse transcriptase (hTERT): Catalytic subunit

2. Human telomerase-associated RNA (hTR): RNA subunit that acts as a template for hTERT

Reintroducing hTERT prevents entrance into crisis

• Northern blot Assay shows that hTERT is highly present in immortal cells

What techniques can be used to test the role of telomerase?

1. Southern blot that shows HEK cells (with no telmerase) infected with control or hTERT. Those infected with hTERT should see an increase in the telomere length.

2. Graph that shows HEk cells with hTERT enter crisis much later than those without

3. Expression of antisense RNA in the telomerase HeLa cells causes them to stop growing after 23 to 26 days

4. Expression of DN hTERT subunit in telomerase (+) human tumor cell lines cause them to lose all detectable telomerase activity, and, with some delay, to enter crisis.