2. Week 1: Cell Cycle (copy)

Lecture 2 - 643 - Faridi

What are the categories of cells that are in vivo (3)

• Cells that highly specialized and lack the ability to divide, once differentiated

  • Neurons, muscle cells, RBCs

• Cells that do not divide normally but can cycle with an appropriate stimulus

  • Liver cells (surgery)

  • Lymphocytes (antigen interaction)

• Highly mitotic cells - these are constantly dividing

  • Male gametes

  • Hematopoietic stem cells

  • Epithelial cells

Recall the overall pathway from growth factor binding to cell response

• Growth factors and RTKs

  • Growth factors bind to receptor tyrosine kinases (RTKs)

  • RTKs are typically monomers before binding

  • Ligand binding causes:

    • Dimerization

    • Trans-autophosphylation

• Key concept: Ligand binding

  • growth factor = ligand

  • ligand binds outside the cell (in this image ligand does not enter cell - signal transduction)

  • triggers signal transduction inside the cell according to image

• Signal transduction

  • activates mitogenic signals - mitogenic is growth-promoting signals

  • these signals involve intracellular biochemical processes

  • leads to cell cycle activation

  • cell cycle = process of cell growth and division

What is cancer

• arises through a series of somatic alterations in DNA that result in unrestrained cellular proliferation

Recall the eukaryotic cell cycle - what are the phases involved?

• Interphase - normal cell function, and then the cell divides into the mitotic phase

• In the Mitotic phase includes

  • Mitosis (DNA division) and Cytokinesis (division of cytoplasm)

Recall the processes of Interphase

• Gap/growth phase “nothing happening”

  • G1 - a lot of cell growth

    • 2N (N = # of genes per chromosome) - cell starts as 2N and begins to grow

• S phase (synthesis)

  • begins with DNA synthesis, and it ends when the DNA is fully replicated; more growth is occuring within this cell

  • at the end of S-phase, 2N has doubled to 4N - doubled the amount of DNA, getting ready for the cell to dividde

• G2

  • gets materials ready for cell division

• G0 - typically ignored, not seen in image

  • this is what neurons are undergoing - have left the cell cycle; these are NOT cycling cells

What are the 4 phases of the M phase? What is the 5th phase? When does the 5th phase begin and what does it form?

• The cleavage furrow is important in cytokinesis because it is the structure that helps divide one cell into two daughter cells.

  • Completes the final step of cell division after mitosis

List the DNA damaging agents and its pharmacological class (3)

• Cyclophosphamide

  • type of alkylating agent

  • damages cells DNA and may kill cancer cells

  • may also lower body’s immune response

• Doxorubicin

  • type of anthracycline antibiotic

  • type of topoisomerase inhibitor

• Cisplatin

  • type of platinum analogue

Identify DNA inhibiting/division agents and its pharm class

• Fluorouracil

  • type of antimetabolite

  • stops cells from making DNA and may kill cancer cells

• Methotrexate (Trexall, Rheumatrex)

  • antimetabolite and antifolate

  • stops cells from using folic acid to make DNA and may kill cancer cells

• Gemcitabine

  • antimetabolite

  • stops cells from making DNA and may kill cancer cells

Identify DNA inhibiting/division agents

What are the key regulators of the cell cycle (2)

• main way is through this protein complex

  • cyclin-cdk complex

    • which is a heterodimeric protein kinases

      • cyclin

      • cyclin-dependent protein kinases (cdk)

  • it’s function is that it controls the timing of the cell cycle

Explain what each member of the heterodimer complex does (the Cyclin-cdk complex)

• Cyclin = the regulatory subunit

  • Its levels go up and down during the cell cycle.

  • It controls when the complex becomes active.

  • Different cyclins appear at different stages of the cell cycle.

• CDK (cyclin-dependent kinase) = the catalytic subunit

  • It is an enzyme (a kinase) that adds phosphate groups to proteins.

  • CDKs are usually present in the cell all the time, but they are inactive alone.

  • They become active only when bound to the correct cyclin.

• Together:

  • Cyclin binds CDK → forms the cyclin-CDK complex

  • The activated complex phosphorylates target proteins

  • This pushes the cell into the next stage of the cell cycle

• So in simple terms:

  • Cyclin = the “on/off timing controller”

  • CDK = the “worker enzyme” that carries out the action

What individuals cyclins and cdks pair up and what is each of their functions?

Where can the restriction point be found? - what cyclins and kinases pair up to move past this?

• paring between cyclin and cdk is very specific

  • remember if cells are not cycling, they are referred to as G0

  • G1

    • Cyclin D binds to CDK6 and drives/allows for it to go past the restriction point and move into the next phase

    • Cyclin E and Cyclin A will bind to CDK2 - initiates DNA synthesis in early S phase

    • Cyclin B and A bind to CDK1 to transition from G2 into M

• restrict point is somewhere in late G1 - example of a cell cycle “check point”

What factors regulate CDKs in order to activate the cyclin-cdk complex?

1. CDK concentrations do not change, Cyclin Concentrations is what changes

   1. cyclin concentration is the first factor that comes into play

2. CDK has two phosphorylation sites

   1. 1st site - tyrosine (tyr) 15 - if there is a Phosphate (P) here, it inhibits the activity of CDK

      1. the Phosphate gets here by a kinase → wee1 (yeast)

         1. a phosphatase is an enzyme that is used to remove the tyr 15, more specifically cdc25 phosphatase allowing for cdk activation

   2. 2nd site - threonine (thr) 160 - Phosphate here allows for cdk activity that is added by CAK

      1. cdk7 is a specific CAK

Cell cycle regulation in total

1. no cyclin present, so cdk is inactive

2. stimulation

3. still inactive tyr-15 inactives the complex, must be removed by a phosphatase (cdc25) to remove the P on tyr-15

4. still inactive because the P in tyr-15 is still not removed

5. active since P on thy-160 and there is no P on tyr-15

What factors regulate CDKs after the cyclin-cdk complex has been activated?

3. CKIs - these are endogenous proteins within a cell

   1. a CKI blocks cdk activity in response to certain things listed in the image

   2. why would we want the cell cycle to be off?

      1. because not all cells should be dividing

      2. overgrowth/contact example: in hepatocytes when cells are touching their neighbors and there’s no space left, they stop dividing = CKIs increase and cell cycle stops

      3. DNA damage example: in DNA damage such as sun exposure, do not want damaged cells copying themselves, so CKIs increase and cell cycle stops

   3. CKI levels very high in neuronal cells, not dividing cells

   4. Understand what CDK inhibitory proteins (cip or kip) and INK4 proteins do

      1. p16

      2. p21

Mechanism of action of CDK4/6 inhibitors in breast cancer

• used in certain types of breast cancer like HR-positive, HER2-negative breast cancer

• when CDK4/6 is inhibited, this allows for Rb to stay active/”on” so that cancer cells cannot go past the restriction checkpoint from G1 to S phase

  • Rb is a tumor suppressor protein

Cyclin-dependent kinase (CDK) inhibitor agents

• Palbociclib (Ibrance)

  • type of CKD inhibitor

  • blocks certain proteins, which may help keep cancer cells from growing

  • Ribociclib (Kisqali)

  • Abemaciclib (Verzenio)

Examples of antiestrogens and estrogen modulators

• Tamoxifen

  • antiestrogen (selective estrogen receptor modulator, SERM)

  • Fulvestrant (pure antiestrogen)

    • binds to proteins and blocks estrogen receptors

• Anastrazole (Arimidex)

  • aromatase inhibitor

  • lowers amt of estrogen made by the body

Examples of inhibitors of mitogenic receptors or pathways

• Trastuzumab

  • Monoclonal antibody and type of HER2 receptor antagonist

  • binds to a protein called HER2, which is found on some cancer cells

• Erlotinib

  • type of EGFR tyrosine kinase inhibitor (TKI)

  • blocks protein called EGFR

• Idelalisib

  • type of PI3K tyrosine kinase inhibitor

  • blockers certain proteins

What factors regulate CDKs - what happens when cyclin (and CKI) concentrations decrease?

• recall the first 3 cell cycle regulation factors

4. it is targeted for degradation when cyclin concentrations go down by Ubiquitin-proteasome pathway

   1. know the three initiating proteins and that they initiate the process of cyclin or CKI degradation for specific cyclins

   2. Ubiquitin ligases include E1, E2, E3 - come and puts ubiquitin on

   3. once ubiquitin molecules made, the proteasome recognizes this and degrades cyclin and/or CKI

Structure of the proteasome

• green circular structure - Ub

• yellow structure - cyclin

• illustrates how cyclins are degraded

• knowing this, can we therapeutically target cancers where too much degradation is occurring?

  • yes, in myeloma cancer - too much degradation

    • use agents that target this

Bortezomib (Velcade, PS-341)

• What happens when the proteasome is blocked?

• Proteins accumulate inside the cancer cell:

  • damaged/misfolded proteins

  • pro-apoptotic proteins

  • cell-cycle inhibitors

• This creates severe cellular stress.

• Result: cell cycle arrest, unfolded protein response, apoptosis (cell death)

define the significance of checkpoints

What are the types of checkpoints