L17 regulation of cell growth (CC3)

• if m-cyclin is not degraded, then anaphase arrest occurs.

  
• if APC (E3 ligase that leads to degradation of m-cyclin) is blocked by an inhibitor, there is metaphase arrest because the cohesin complex is not destroyed by separase

  
• cohesins are proteins that have DNA binding domains, ATP binding domains, coiled coiled domains and a hinge. cohesins cross-link two adjacent sister chromatids, gluing them together. condensin is a cohesin dimer. condensin condenses the DNA and is responsible for forming chromosomes.
• sister-chromatid separation by APC:

  1. APC becomes active with cdc20 binding.
  2. securin (inhibitory to separase) and separase are bound together. when securin is removed, separase (protease) becomes active. securin is then ubiquitinylated and degraded.
  3. separase destroys the cohesin complex allowing cell to go from metaphase to anaphase.
• m-cyclin and s-cyclin have to be removed in order for the cell to undergo cytokenesis
• cell has a mechanism to stop the separation process if not all chromosomes are attached. mad2 protein will attach and signal to the cell to not go through metaphase and anaphase if not all chromosomes are attached
• how to ensure that m-cyclin levels go down after m-phase:

  cells that have no G1 and G2 (go through M and S phase after fertilization) have cdc20 active during late m-phase, allowing APC to be active and degrade the m-cyclin. this is the mechanism for embryonic stem cells
• how to ensure that m-cyclin levels go down after m-phase:

  in cells with a G1 phase, they have a second activator for APC called Hct1 along with cdc20. this additional activator allows APC to stay active, allowing m-cyclin to stay low during G1 (we don't want active cdk complex in G1 because the cell is resting during this phase)
• in cells with no G1 phase, the cdc20-APC activity will decrease as the cell goes into s phase. in cells with G1 phase, the cdc20-APC activity will decrease as the cell goes into G1, but the Hct1-APC activity will increase during G1.
• another mechanism for controlling m-cdk activity during G1 in cells with a G1 phase is by activating another complex called Sic1 (in yeast) or p27 (in mammalian cells). this complex is active as the same time as the Hct1-APC complex. these two combined lead to inactive M-cdk.
• during G1, when G1-cdk becomes activated, it will activate the G1/S-cdk complex. this complex will inactivate Hct1 and Sic1 by phosphorylating them. then, this will trigger the activation of s-cdk.
• G1-cdk is overproduced in cancer cells. many patients with severe cancer will have excess G1-cdk, meaning they will get a G1-cdk inhibitor as well as chemotherapy as thier treatment
• mechanism to control s-phase:

  1. Rb protein is activated, which leads to an inactivated e2f (transcription factor for DNA replication - S phase).
  2. active G1-cdk receives signal to promote s phase leading to its accumulation. active G1-cdk will phosphorylate Rb protein, leading to active e2F.
  3. e2F will transcribe genes needed for S phase, including G1/S cyclin and S cyclin. This creates an active S-cdk complex, and official entering of S phase
• the mechanism to control S phase is a positive feedback loop because the active S-cdk will further enhance phosphorylation of Rb, which leads to more active e2F and more active S-cdk. active e2F is also a positive feedback loop because it will transcribe more of itself when its active
• Rb protein stands for retinoblastoma protein. this disease results from a mutated Rb gene and a normal one. if the remaining copy gets mutated, then e2F will no longer be inhibited, meaning the cell will always be in s-phase, resulting in a tumor.
• the triggering mechanism into s-phase pathways is always mutated in any cancer cell
• in a normal cell, as nutrition of the cell decreases, the time of the division cycle increases (the cell needs more time), but if nutrition is completely taken away, then the cell will stop growing.
• in a mutant cell, if nutrition is reduced, the cell itself will get smaller and smaller (but they keep dividing!!). this is a cell without nutritional cell-cycle control
• model of how yeast may coordinate cell growth and cell-cycle progression:

  1. as the cell grows in size, there is more G1 cyclin (Cln3) produced that binds to a protein bound to DNA
  2. there reaches a point where all the Cln3 (G1 cyclin) is bound to the proteins bound to DNA, and so free G1 cyclin is left to bind with cdk to trigger the next cell cycle
• DNA damage will arrest the cell cycle in G1. usually, mdm2 is bound to p53 which promotes p53 degradation via the proteasome.
• when there is damage in the cell, p53 becomes phosphorylated, and this blocks p53 binding to Mdm2. p53 will then accumulate and trigger transcription of p21, a CKI (cdk-inhibitor protein). p21 will then clamp around an active G1/S-cdk or an active S-cdk and make it inactive. this pauses the cell in G1 and gives time for the DNA damage to be repaired
• p53 is a tumor suppressor protein. when it is missing or mutated, the cell will continue to grow (because the cell stays in G1 forever due to p21 over cdk). this protein is missing or deleted in half of all human cancers
• if p53 is gone by mutation, the cells will go into S phase with damaged DNA and produce mutant cells
• apoptosis (cell death) can be useful in normal development (like formation of fingers or tail of a frog coming off).
• in apoptosis, cell death is controlled, there all the intracellular contents are degraded. in necrosis, the cell contents are spilled out, and this is a very dangerous process.
• mitogens are growth factors in a cell, they are small molecules. they regulate cell proliferation (cell division). they do this by binding to a transmembrane receptor which signals the intracellular Ras. Ras signals to MAP kinase, which passes the cytosol into the nucleus to activate transcription of myc. myc, through three different pathways, induces entry into S phase
• mitogen stimulates receptor which stimulates ras which stimulates creation of myc (a transcription factor). myc then induces transcription of

  • cyclin D, which increases G1-cdk activation, RB phosphorylation and increased E2F activity to enter into S phase
  • SCF subunit, increases p27 degradation, G1/S-cdk is activated, Rb is phosphorylated, increasing E2F activity to enter into S phase
  • E2F gene directly which increases amount of E2F activity allowing entry into S phase
• a cell may receive too much mitogen when another cell dies by necrosis or other events happen in the cell. when there is too much mitogen, Myc will create p19ARF which binds to Mdm2 and prevents it from bringing to p53. active p53 can cause cell-cycle arrest or apoptosis of the cell. in many cancer cells, p19ARF is deleted leading to cells growing forever
• we immortalize cells if we remove p53 or insert telomerase
• normal human cells lack telomerase, a protein that puts buffer regions at the end of chromosomes to protect the chromosome from DNA damage. cells lacking telomerase will divide about 50 times and then as DNA gets shorter and shorter, p53 will stop the cell divisions. cells that have telomerase will divide 70-80 times.
• another way in which growth factors promote cell growth is through stimulation of PI 3-kinase, promoting eIF4E and phosphorylated S6 kinase, increasing mRNA translation and cell growth.
• cell growth (PI 3-kinase) and proliferation (mitogens) are usually coupled processes. however, sometimes they are uncoupled as in a lymphocyte (small neuron) where nerve growth factor will stimulate only cell growth and not cell division
• without survival factors, the cell will die.
• survival factors are molecules, similar to growth factors. they signal a receptor that phosphorylated protein kinase B (making it active). from here, the PKB will either (1) inactive Bad (a protein) creating an active Bcl-2 supressing apoptosis (2) phosphorylating (making inactive) a gene regulatory protein that when active leads to apoptosis
• when cells are put in petri dish with a liquid that has growth factors, amino acids, survival factors, etc. then the cells will start to replicate. when all of these molecules have been used up, the cells will create a confluent monolayer and no longer proliferate. cancer cells with this same medium will lead to a 3D growth which is dangerous and abnormal
• normal cells need anchorage in order to grow (except for cells in blood stream that naturally float). in normal cells, without attachment, they will die
• many cancer cells will be able to float (not adhere to anything) and be able to proliferate and travel
• • three factors important for cells to survive, grow and proliferate
  • mitogens (involved in cell proliferation)
  • growth factors (involved in cell growth) - NGF for ex
  • survival factors (tell the cell to survive and not undergo apoptosis)