1 & 2- Kearns - Cell Cycle Regulation and Gene Expression

Overall, disruptions in the cell cycle result in _______________.

cancer

What are cell cycle non-specific disruptions?

• abnormal cell division rate

• dysregulation of progression from one phase to the next

  • could be G1—> S or M—→G1 etc. etc.

All of the following are examples of non-specific cell cycle disruptions except _________.

a. DNA mutations during S phase.

b. the progression from S phase to G2.

c. abnormal cell division rate.

d. the progression from G1 to S phase.

a

What are cell cycle specific disruptions?

• DNA mutations

• DNA replication mistakes

• microtubule assembly disruptions

All of the following refer to disruptions in cell cycle specific regulation in cancer except ____________.

a. DNA mutations

b. disruption in microtubule assembly

c. abnormal cell division

d. mistakes in DNA replication

c

Describe the M phase of the cell cycle:

• “MITOTIC PHASE”

• mitosis and cytokinesis occurs

• cell physically divides here

Describe the Interphase phase of the cell cycle:

• broader term that refers to the G1, S, and G2 phase all together

  • G1- organelle development, growth

  • S- DNA REPLICATION (2n—>4n)

  • G2- safety check genome integrity prior to next division

Between each phase of the cell cycle there are _____________. This is also a drug target in cancer.

checkpoints

What’s the difference between cyclins and cyclin dependent kinases (CDK)? What do these both combine to do?

• CYCLIN- PROTEINS that bind to and activate cyclin dependent kinases

• CYCLIN DEPENDENT KINASES (CDKs)- ENZYMES (special protein) that PHOSPHORYLATE

both of these combine to drive the cell cycle through each phase

True/False: Different cyclins are expressed equally in concentration throughout each stage of the cell cycle.

false- each cyclin fluctuates based on what phase of the cell cycle

Are cyclins anticancer targets? Why or why not?

• Cyclins ARE anticancer targets

• Why? remember CDK+Cyclin= phosphorylate in order to continue the cell cycle, so if we stop this we will DEPHOSPHORYLATE, and we STOP the cell cycle and STOP cell growth!

How do healthy cells maintain their genomes?

tumor suppressor genes stop the formation of tumors

When we say “maintain the genome” what are we referring to?

how a cell maintains 46 chromosomes

Explain how the p53-p21-RB signaling pathway NORMALLY works in response to DNA damage.

1. DNA damage activates p53

2. p53 activates p21

3. p21 inhibits CDK/Cyclin complex= block cell cycle progression

4. no phosphorylation of RB/E2F further prevents cell cycle progression

What are the 2 fates of DNA?

1. Replication (DNA—> DNA)

2. Protein Synthesis (DNA—> RNA —→ Proteins)

Which is the building block of DNA?

a. nucleoside

b. nucleotide

b.

In general, what’s the difference between intramolecular and intermolecular bonds?

• intra= inside the molecule

• inter= between 2 molecules

In DNA, where are intramolecular bonds found and give an example:

• bonds in the BACKBONE

• example—> PHOSPHODIESTER BONDS BETWEEN NUCLEOTIDES

In DNA, where are intermolecular bonds found and give an example:

• bonds between 2 separate strands

• example- Hydrogen bonds between A and T

In cancer, would we want to target intra or intermolecular bonds?

intramolecular

How do we unwind DNA when it’s supercoiled?

TOPOISOMERASE I or II

What is the chemical reaction between Top I and DNA? What bond is formed and between what?

• Summary of steps- “reversible ester bond between a tyrosine on top and the phosphate group on the DNA”

• Detailed Steps-

  • 1.Top binds to the supercoiled DNA

  • 2.The tyrosine on top has an -OH group that acts like a nucleophile and attacks the phosphodiester bond of the DNA backbone

  • 3.This breaks the one strand of DNA and creates a nick that relieves torsional strain

  • 4.To repair the DNA strand the -OH group on the DNA performs a second nucleophilic attack on the phosphotyrosine bond which makes top dissociate

What are the 3 important enzymes involved in DNA replication? What are each of their roles and state whether or not they are drug targets in cancer.

1. TOP- unwinds DNA

2. HELICASE- separates (“unzips”) the 2 DNA strands

3. DNA polymerase- adds nucleotides to the dividing strands

ALL 3 ARE DRUG TARGETS IN CANCER

Nucleotide biosynthesis no matter if it’s a purine or pyrimidine always begins with what reaction? Using what enzyme?

___________ ——> _________?

Ribose-5-P ———> PRPP using PRPS

Explain how guanine is made de novo (not the salvage pathway):

• include which step is folate dependent

• which step is where oxidation occurs?

1. PRPP —> 5-phosphoribosylamine using GPAT

2. 5-phosphoribosylamine to IMP (FOLATE DEPENDENT)

3. IMP—> XMP —> GMP (WE OXIDIZE IMP to GMP)

4. GMP—> GDP using GUANYL KINASE

5. GDP—> dGDP using ribonucleotide reductase

   • reduction of OH to H

Explain how adenine is made de novo (not the salvage pathway):

• include which step is folate dependent

• which step is where aminated occurs?

1. PRPP —> 5-phosphoribosylamine using GPAT

2. 5-phosphoribosylamine to IMP (FOLATE DEPENDENT)

3. IMP—>AMP (WE AMINATE IMP to AMP)

4. AMP—> ADP

5. ADP—> dADP using ribonucleotide reductase

   • reduction of OH to H

Explain how cytosine is made de novo:

1. PRPP+ Orotate—> UMP

2. UMP—> UTP

3. UTP—> CTP (amination reaction)

4. CTP—> dCTP using ribonucleotide reductase

Explain how Thymine is made de novo:

1. PRPP+ Orotate—> UMP

2. UMP—> dUMP using ribonucleotide reductase

3. dUMP —> dTMP using thymidylate synthase (folate dependent)

4. dTMP—> dTTP

What are the primary targets in cancer if we want to inhibit MITOSIS?

microtubules synthesis

What do microtubules bind to? What does this signify?

• bind to the kinetochore of chromosomes

• once this occurs—> cell division WILL OCCUR there is NO GOING BACK

What are some functions of microtubules?

• chromosome segregation in mitosis

• mechanical support (cell movement)

• transport

• intracellular transport

A microtubule is a heterodimer with what kind of symmetry? Explain.

has inherent asymmetry meaning that is has a -alpha terminus and a +b terminus

What would high concentrations of GTP do to microtubules?

• GROW!!!! microtubule lengthens

• rate of breakdown (GTP hydrolysis) = rate of growth (polymerization)

What would low concentrations of GTP do to microtubules?

• SHRINK

• rate of breakdown (GTP hydrolysis) > rate of growth (polymerization)

If we were designing an anti-cancer drug, would we want the drug to depolymerize or polymerize microtubules?

DEPOLYMERIZE

What is gene expression?

“the level to which our proteins are transcribed” (basically how our genes and DNA are used to make proteins)

What’s the difference between RNA polymerase and DNA polymerase?

RNA polymerase—> deals with protein synthesis

DNA polymerase—> deals with DNA replication

What is a transcription factor?

Regulatory proteins that bind to the DNA in the nucleus and effect transcription

The binding of _____ is required for transcription to start.

RNA polymerase

What does RNA polymerase bind to start transcription? Where is this region located?

promoter region, located right before the gene

Upstream of the promoter is a distal regulatory region that contains what 3 regions where transcription factors bind? What is their effect on gene expression?

a. insulator- protects from inappropriate signals

b. silencer- inhibits gene expression

c. enhancer- promotes gene expression

What does the term “Basal level of transcription” mean? What determines this?

• it’s the normal or “base” level of transcription aka the amount of transcription we would have without any of the enhancers/silencers/or anything promoting/inhibiting transcription.

• determined by the promoter sequence

Can RNA polymerase bind to the promoter region on its own?

no, needs TFs

What’s the difference between DNA replication and protein synthesis in terms of…

• goal

• enzymes that are therapeutic targets

• starting nucleotides

• products

• location in the cell where it occurs

DNA is stored in highly structured complexes called…

chromatin

In order for a gene to be transcribed what must happen to chromatin?

make accessible chromatin regions—→ modify/unwind nucleosomes (DNA+histone) in order to make the DNA accessible to RNA polymerase and other TFs

What are epigenetic changes?

By altering the DNA/Chromatin Structure, this affects how easy it is for enzymes needed for transcription to interact with and transcribe genes aka effects GENE EXPRESSION

How are epigenetic changes different from DNA mutations?

DNA mutations change the DNA sequence, but epigenetic changes alter the chromatin (DNA) STRUCTURE

Epigenetic changes may be caused by what?

• development

• environmental changes

• drugs/pharma

• aging

• Diet

Can drugs that treat cancer be an epigenetic factor?

yes

Where in the cell does transcription take place?

nucleus

Where in the cell does translation take place?

cytoplasm/ribosomes

What are the steps in protein synthesis?

1. transcription

   • remove histones and uncoil DNA

   • separate DNA strands

   • assemble nucleotides into a single strand of mRNA using RNA polymerase

2. post-transcription modification

   • mRNA before leaving nucleus needs INTRONS cut out and the remaining exons are spliced together

3. translation

   • codons of mRNA binds to anticodons on tRNA

   • each tRNA carries a specific AA

   • rRNA then strings the AA together

   • Now you have a protein :)

3 mRNA nucleotides are called a_______.

codon

True or False: Introns are the coding sequence of mRNA.

false

What are housekeeping genes?

genes required AT ALL TIMES and are expressed CONTINUOUSLY

Expression of housekeeping genes can be modulated by…

regulatory proteins aka TRANSCRIPTION FACTORS by enhancing or repressing the interaction between RNA polymerase and the promoter

What is a specificity factor?

alter the specificity of RNA polymerase for a given promoter or set of promoters

What is a repressor?

impedes access of RNA polymerase to the promoter

What is an activator?

enhances the RNA polymerase—promoter interaction

Which of the following do epigenetic regulation of gene expression control:

a. TATA sequence

b. promoter region

c. regulatory transcription factors

d. enhancer sequences

c, d (WHY? remember that epigenetic influences how accessible a gene is by messing with the structure, so it would control how TFs bind to these regions)

Which of the following do genetic regulation of gene expression control:

a. TATA sequence

b. promoter region

c. regulatory transcription factors

d. enhancer sequences

a, b (WHY? genetic regulation refers to the DNA sequence itself and the TATA sequence and promoter region are actual parts of the gene)

mRNA synthesize is regulated by phosphorylation and dephosphorylation. What are 2 methods of phosphorylation and dephosphorylation? What are the enzymes involved?

1. protein phosphorylation

   • protein kinase= phosphorylate

   • protein phosphatases= dephosphorylate

2. GTP-binding phosphorylation

   • GTP binding= phosphorylate

   • GTP hydrolysis= dephosphorylate

What are the 3 steps of translation?

1. initiation- mRNA binds to P site, tRNA binds to P site carrying an AA

2. elongation- the tRNA moving from the P site moves it’s AA to the tRNA on the A site and then that tRNA moves from the P site to the E site and is released

3. termination- stop codon on mRNA, new polypeptide