Cancer Biology Exam 2 Material

Cell cycle control is important for the cell to make the decision whether to "proliferate or not." Extracellular signals such as growth factors and the extracellular membrane serve as cell cycle control proteins that tell the cell to proliferate or not to proliferate.

G0 = Not actively proliferating; Quiescent. Some cells are permanently in G0 such as neurons. Some cells are transiently in G0. If these cells are given the proper signals, they may divide again.G1 = Decision making phase, considering extracellular signals. The R Point (Restriction Point) is the point of no return; The point at which the cell decides to divide. After this, the cell will divide (or die if things get messed up).S = Synthesis; DNA replication.G2 = 3-5 hours; Not exactly sure what is happening here. Maybe cell is preparing for mitosis.M = Mitosis. Approximately 1 hour. Prophase, Metaphase, Anaphase, Telophase.

Normally, checkpoints throughout the cell cycle make sure everything is okay. If not, the cell undergoes apoptosis. Cancer cells blow past checkpoints.

A CDK is a serine/threonine kinase. CDK needs cyclin in order to be active. Each cell cycle phase has a different cyclin-CDK. CDK protein levels are constant overall. Individual CDK protein levels fluctuate depending on the phase of the cell cycle. The drop off in cyclin levels is mediated by ubiquitination and degradation.

*Rb (at R point).*Proteins needed for DNA replication (S Phase).*Histones: Phosphorylation state will regulate DNA packaging.*Nuclear membrane proteins get phosphorylated and then degraded by the proteasome.

Cyclin D pairs with CDK 4 and CDK 6. Cyclin D protein levels fluctuate based on extracellular signals during G1. Signaling pathways that lead to proliferation do so by regulating Cyclin D protein levels (ex. integrins and Ras).

Mitogen (GF)-------->Receptor-------->Ras-------->Cyclin D------->Cell Cycle.

Inhibitors bind CDKs and inhibit their kinase activity. They bring them to the cytoplasm so they can't regulate the cell cycle.*P15 inhibits Cyclin D-CDK 4/6. P15 will be expressed downstream of TGF beta signaling. TGF beta is an anti-growth factor. P15 inhibits kinase activity of CDK 4/6---> does not enter cell cycle.*P21/P27 are activated by DNA damage. They don't inhibit Cyclin D-CDK 4/6.

Phosphorylation status changes with the cell cycle. Phosphorylation is controlled by cyclin-CDKs.*G1--Hypophosphorylated (few phosphate groups).*Past R Point--Hyperphosphorylated (a lot of phosphate groups).*End of mitosis--Dephosphorylated (PP1 takes phosphate groups off).

Rb binds to a transcription factor called E2F. E2F is a transcription factor for genes needed for profliferation such as RNAP and cyclins.When Rb is dephosphorylated, it binds E2F and inhibits transcription.Whn Rb is hyperphosphorylated, it can't bind to E2F so E2F can act as a transcription factor.

Unphosphorylated Rb binds to E2F. Prevents E2F from transcribing DNA.Mitogens such as Growth Factor increase Cyclin D levels. Then Cyclin D/CDK4/6 hypophosphorylate Rb. Rb is still bound to E2F. The process reverses if there is no Growth Factor.Cyclin E/CDK 2 hyperphosphorylates Rb (it can only act on hypophosphorylated Rb). It can act on multiple Rbs. This process occurs past the R point. Rb can no longer bind to E2F so E2F is able to transcribe many things including more Cyclin E. The increased transcription of Cyclin E leads to positive feedback. Rb keeps getting phosphorylated.

To turn on transcription factor, E2F must recruit RNAP (if DNA is tightly packaged in chromosomes it can't be transcribed). Therefore, E2F will also recruit other proteins that regulate DNA packaging (open it up).Rb binding to E2F prevents E2F from interacting with other proteins.

Cyclin E-CDK 2 complexes phosphorylate P27 and then P27 is ubiquitinated and rapidly degraded by the proteasome. P27 can't inhibit cyclin E-CDK 2 anymore.The destruction of P27 activates more Cyclin E-CDK 2 complexes that proceed to phosphorylate and inactivate P27 molecules.

Transfect epithelial cells with HPV genes. Do the genes cause the cells to transform? Yes, E6 and E7 genes transform cells.How do E6 and E7 cells cause cancer? Stain cells with fluorescent molecules, use flow cytometry. More DNA = More fluorescence. Varies with stage of the cell cycle. Then irradiate cells. Irradiation can cause damage and allows you to see if cell cycle has already been messed up. Cells transformed with E6 and E7 indicated messed up cell cycle (cell cycle was stalled, stuck in mitosis). Irradiated normal cells look the same as non-irradiated normal cells so the cell cycle was not messed up.How are the cells responding to irradiation? Normal cell: Rb is not phosphorylated after 24 hours = Exit cell cycle. E6: No P53. Cell cycle is messed up but Rb is present so results look similar to normal cell. E7: No Rb. Can't stop going through the cell cycle.How is E7 regulating Rb? E7 is causing Rb degradation (yes according to Western blot). E7 is causing change in transcription of Rb (not the case according to Northern blot).

SV40 causes cancer in mice because cellular protein P53 is interacting with a viral protein.In order to find out what P53 does, a mutated P53 gene was taken from mouse cells and put into other mouse cells to overexpress P53 and these cells became cancerous. Researchers did not realize right away that this was a mutated version of P53. Original conclusion: P53 is an oncogene. But wait! This is a mutated P53 gene. P53 is actually a tumor suppressor gene.