Lecture 11

Question 1

Mutations in TS (tumor suppressor genes) can lead to:

Answer 1

1. Defects in DNA repair
2. Loss of cell cycle checkpoints
3. Evade apoptosis

Question 2

What are proto-oncogenes?

Answer 2

These are normal genes found in our cells located on chromosomes that help function with growth + signaling

Question 3

How do oncogenes occur?

Answer 3

Oncogenes occur when proto-oncogenes mutate into oncogenes that can be cancer causative mutations
- Oncogenes/mutations cause loss of growth control
- They also often have dominant phenotypes
- Often form as de novo somatic mutations (not inherited)

Important notes about oncogenes:
*miRNAs can also function as oncogenes
*RAS is a common oncogene found in cancer
- Happens as a point mutation in the protein-coding region that turns RAS gene ON all the time, losing its regulation, making it insensitive to growth factors or extracellular signals

Question 4

Oncogenes allow for:

Answer 4

1. Cell growth in the absence of signals
2. Insensitivity to antigrowth signals

Question 5

What are the hallmarks of cancer?

Answer 5

The hallmarks of cancer are the biological capabilities that cancer cells acquire as they develop into tumors.

There are 6 of them:
1. Self-sufficiency in growth signals
- Cancer cells generate their own growth signals so they don’t need an external stimulus to continue to grow
2. Insensitivity to antigrowth signals
- Can evade signals that to a normal cell, would actually push out of the cell cycle
3. Tissue invasion and metastasis
- Certain cells are under + selection to promote cancer growth and tumor formation
4. Limitless replicative potential
- Activating telomerase (ON) can occur to overcome the crisis stage.
- Telomeres are added to the ends of chromosomes every cell division to they keep on dividing with no limit
- Telomerase is an enzyme made up of RNA and proteins that adds telomere repeats on ends of chromosomes.
5. Sustained angiogenesis
6. Evading apoptosis

Question 6

In what cells can you find an activated telomerase?

Answer 6

• Embryonic cells
• Stem cells
• Cancer cells

Question 7

Describe the multi-step development of colon cancer

Answer 7

It is a multi-step process to get to the point of having a tumor with angiogenesis (formation of new blood vessels from existing ones). This happens from a number of mutations, NOT just one mutation.

For colon cancer, we see:
1. Loss of mutation of APC TS gene in normal epithelium
2.DNA methylation in hyperproliferative epithelium
3. Activation of KRAS oncogene (oncogene mutation) in early adenoma
4. Loss or mutation of TS gene on 18q in intermediate adenoma
5. Loss or mutation of TP53 in TS gene

Question 8

How can gene expression get activated?

Answer 8

Loss of methylation (hypomethylation) at promoters.
- In normally silenced genes, will see DNA methylation at the promoter.

Question 9

Why are there different cancer cell types in tumors?

Answer 9

The different cell types can be from various mutations that formed.
- There tends to be a + selection for the mutations that help to increase the likelihood of being beneficial to the tumor, so that it may persist.

Question 10

Describe driver mutations

Answer 10

These are usually seen in oncogenes and TS genes

• These mutations want to divide as much as possible to allow for the continuation of carcinogenesis, metastasis, ultimately and possibly leading to death.

Question 11

Describe passenger mutations:

Answer 11

These have no consequential effects

Question 12

What is NF1?

Answer 12

NF1 is a tumor suppressor gene that produces a protein called neuorfibrin that prevents cells from dividing too rapidly

Question 13

Describe the Philadelphia chromosome

Answer 13

• A well known translocation that results in a gene fusion of the BCR and ABL1 genes, making an oncogene by activation.
  
  • Causes myelogenous leukemia
    
    • ABL1 gene will become expressed in B-cells as an oncogene after gene fusion.

Question 14

Describe the BCR gene

Answer 14

BCR gene encodes the B-cell receptor protein that is expressed in B-cells
- B cells are a part of the immune system that surveys blood to find potential pathogens; the expression of the BCR gene is a part of their immune function

Question 15

Describe the ABL1 genes

Answer 15

ABL1 is a proton-oncogene & tyrosine kinase (adds phosphate group to tyrosines in a.a. sequence of some other proteins

Question 16

What are the 4 ways of activating proto-oncogenes?

Answer 16

1. Amplification
   
   • gain of function and is turned on permanently, -> amplifies production or their activity
   • Usually amplification of CNVs, promoter mutations, SVs (ex: Philadelphia chr.)
2. Point mutation or small intragenic deletion
   
   • Ex: RAS, a gene that encodes for the protein, GTPase, a mutation can happen at active site of the GTPase, allowing it to always be ON in the GTP-bound state.
   • It no longer allows for the hydrolization of GTP –> GDP to turn it OFF
3. Chromosomal rearrangement by creating a novel chimeric gene
4. Chromosomal rearrangement by placing a gene under the control of a powerful enhancer

Question 17

Describe the pathways that can lead to heterozygosity

Answer 17

1. Homologous recombination: between the locus + centromere
   
   • there is a crossover that occurs between 2 homologous chrs. between the mutation of interest + the centromere
   • can lead to reduction in homozygosity or retention in heterozygosity after mitotic segregation of sister chromatids into their daughter cells
2. Chromosome loss
   
   • lose chr. that carries the good copy of the allele via non-disjunction – failing to divide equally leading to elimination or duplication in daughter cells and thus loss of heterozygosity
   • Ex: Bb –> BB or bb

Question 18

What is loss of heterozygosity?

Answer 18

Start out with -/+ and end up with a homozygous for the mutated allele

Question 19

What are Tumor Suppressor (TS) genes and what is their function?

Answer 19

• Normal cell function: to protect cells by regulating cell division and prevent excessive cell growth
  
  • This is done by slowing down cell division and apoptosis
• A TS gene typically requires a loss of function of both alleles to be recessive at a cellular level and cause cancer
  
  • TS genes are ESSENTIAL to grow as an embryo and so they are not homozygous in which the embryo is not viable.

Question 20

What are the different types of TS genes?

Answer 20

There are 3 different types:

1. DNA damage repair genes
   
   • encodes for DNA repair proteins
2. Cell cycle regulation
3. Apoptosis (last resort)
   
   • Genetically programmed cell death is imposed by the cell itself because it sense too much stress or damage.

Question 21

What is p53?

Answer 21

p53 is called the “guardian of the genome” which functions to stop at the breaks in the cell cycle.
- It is the most mutated TS gene in most cancers
- It hals cell division to repair damage and if damage is not repaired, it will undergo cell death
- When activated, it will activate CDKNA1 which will stop DNA replication/cell division

Question 22

What is ATM?

Answer 22

ATM is activates when there is a DNA damage response such as:
1. irreparable damage
2. repairable damage

Question 23

What occurs with p53?

Answer 23

• ATM, a kinase, activates p53 via phosphorylation to arrest cell growth
  
  • the phosphate group helps to stabilize the complex –> NOT allowing for cell division

When there is NO mutation, p53 is modified differently:
- instead it will be labeled with ubiquitin by MDM2 which will tell cells to degrade the p53 protein by hydrolyzing it–> getting
rid of it –> allow to continuation of cellular division

In summary:
- No/Mutation p53 = no breaks in cell cycle
- p53 present/stabilized = halts cell devision to repair damage, if it can’t, apoptosis.

*p53 CANNOT be homozygous or else not viable embryo due to being a homozygous recessive mutant

Question 24

Why are checkpoints so important?

Answer 24

Checkpoints are an essential mechanism that ensures the coordination of repair and cell cycle progression.

Question 25

Describe pRb

Answer 25

pRb (retinoblastoma protein) is a key regulator of progression through G1 phase to the S phase
- It is essential in halting the cell cycle/DNA replication when there is a DNA damage response.
- W/O pRb, cell will fail to rest the cell cycle and repair DNA which can lead to cancer

• Mutations can cause retinoblastoma
• When pRb is activated (NOT phosphorylated), it binds to/inhibits E2F from proceeding with transcription thru G1 phase –> cell cycle is stopped
• When pRb is phosphorylated/inactivated/or absent it cannot properly regulate E2F –> allowing for progression of the cell cycle in an uncontrolled manner.

Question 26

What does HPV target?

Answer 26

• Mucosal Squamous Epithelial cells
• HPV expresses certain proteins that inactivate pRb and p53
  
  • E6 and E7 works to turn off TS proteins: p53 and pRb + degrade them

*E6 – binds + degrades p53
- w/out p53, cells can divide with damage because it cannot respond to DDR (DNA damage response) thus also leading to decreased apoptosis

*E7 – binds + degrades pRb
- degrading pRb will allow for the activation of E2F and DNA replication to continue – doesn’t get halted.

• This allows cells to divide with damage; decreased apoptosis.

Question 27

Describe autosomal dominance and give an example

Answer 27

Autosomal dominant mutations can lead to predispositions in cancer.
- Heterozygotes are affected in every generation

Ex: Li-Fraumeni Syndrome
- Mutation in gene TP53 (which is the gene for protein p53)
- Females who have this syndrome generally have nearly 100% chance of developing breast cancer.
- All of those who have this syndrome have a 90% chance of developing 1 or more types of cancer in lifetime and 50% chance of developing before age 30.

*p53 CANNOT be homozygous or else not viable embryo due to being a homozygous recessive mutant

Question 28

What is the Hayflick limit?

Answer 28

This is a phenomenon that happens in normal somatic cells in which there is a limit to the number of times it can divide due to the shortening of telomeres —> which eventually can lead to the state of senescence (no longer divides but still active metabolically)
- Eventually, if telomeres get too short, it leads to the state of crisis which usually leads to apoptosis

*Mutations in the senescence and crisis stages can result in telomerase to be turned on thus making the cells immortal –> recovering cells from crisis and allows for the cell to keep dividing with no limit

Question 29

When does BBF (Bridge Breakage Fusion) occur?

Answer 29

When a cell reaches crisis.
- A cell reaches crisis after senescence when telomeres get so short then the cell is no longer functionally active and typically goes thru apoptosis.

• When crisis occurs, telomeres get so short that they are NO longer protected by the shelterin complex –> this leads to the ends of the chrs. to look like damaged DNA –> this causes the ends to the chrs. to fuse together creating an anaphase bridge in which the ends are covalently linked to each other.
  
  • This anaphase bridge will eventually break and go through the cell cycle –> this gives karyotypes with lots of translocation and aneuploidies which can be seen if individual chrs. are labeled with a fluorescent probe (Spectral Karyotype)

Question 30

How does spectral karyotyping work?

Answer 30

A process called Flow sorting of chromosomes:
*Done in the metaphase state because it is the most condensed state.
- Can treat with Necatozol to depolymerize the microtubules and condense into metaphase state.
- This helps to sync the cells into the same state

1. Separation methods are done in little test tubes to get individual chromosomes
2. Fluorophores are used to paint the individual chromosomes with labeled DNA probes
   - chr. is a template to create new DNA strand with fluorescently labeled nt
3. Probe is hybridized to spread metaphase chrs. on glass slides by first melting the DNA at a high temp and letting it hybridize with the probe when it cools down.
4. Visualize with epifluorescence microscope
5. Classify the chromosomes by colors

Allows to see:
- Aneuploidy
- Translocations
- CNVs (copy number variants)

Question 31

What is chromothripsis?

Answer 31

Chromothripsis (chromosome shattering) is a single event that can lead to cancer as well as other diseases

Occurs due to:
-both strands of the DNA have double strand breaks or there is a cluster of DNA DSBs
- Chrs. will shatter and break into many pieces and get resembled by NHEJ but they are put together in RANDOM order and orientation —> resulting in derivatives of these chrs. and some fragments may be lost to the cell as well.

Question 32

What are the mechanisms of chromothropsis?

Answer 32

There are 3 mechanisms:

1. Chrs. during anaphase do NOT segregate properly
   
   • See anaphase lagging chrs. with no spindle so it just floats around
   • Nuclear envelope breaks down and then reforms to result in a micronuclei formation in which the nuclear envelope reformed around the orphaned chromosome –> chromothripsis (chr. is broken into many pieces and reformed in the envelope)
   • It is then included in the next cell cycle
2. Telomere erosion and dicentric chr. formation
   
   • This leads to the BFB cycle (Breakage-fusion-bridge)
3. Abortive apoptosis due to causing cells tress and chrs. rearrange
   
   • leading to TP53 mutation

NOTE:
*Cell does NOT like micronuclei

Question 33

HeLa cells

Answer 33

HeLa cells was named after the first immortal cell line discovered in 1951 from a woman named Henrietta Lacks who had cervical cancer
- The gynecologist Dr. Howard Jones discovered her tumor at the time and took a biopsy and discovered her cells were immortal and called them “HeLa cells” and they have been used to study many different effects of toxins, drugs, hormones and viruses on cancer cell growth without experimenting on humans.
- Radiation and poisons, human genome, and polio and COVID-19 vaccines were developed using these cells as well!