Cancer Pathways, Apoptosis, and Cell Differentiation Flashcards
P16 Pathway
P16 inhibits Cyclin D and CDK4/6, that inhibition will inhibit the phosphorylation of Rb, which will inhibit E2F.
E2F activation
Leads to proliferation, but also to activation of ARF, which inhibits MDM2, leading to the upregulation of p53. This is a feedback mechanism.
Loss of APC
Causes Beta catenin to be available in the cell, increasing cyclin D and myc.
Myc
Increases the activity of ARF, but also promotes cell proliferation
PI3K Pathway
PI3K turns PIP2 into PIP3. PIP3 activates AKT, which promotes cell survival. PTEN inhibits PI3K.
AKT
Promotes cell survival, activated by PIP3
PTEN
Inhibits PI3K, which prevents PIP3-induced activation of AKT. Thus, PTEN causes apoptosis.
In cancer, PTEN can be mutated causes increased tumor survival
Multi-step model of tumorigenesis
Normal epithelium, (loss of APC) hyperplastic epithelium, early adenoma, intermediate adenoma, late adenoma, (loss of p53) carcinoma, invasion and metastasis.
CDKN2A Locus
INK4A gene encodes both P16 and ARF. Although these proteins share exons 2 and 3, they have their own promoter and first exon. Mutations that affect exons 2 and 3 affect both P16 and ARF.
Transition from epithelial cells to cancer cells
Cell-cell junctions lost (cadherins), loss of polarity, migration increased.
Steps of metastasis
Angiogenesis (FGF and VEGF)
Invasion - Tumor cells attach to ecm, proteases degrade matrix.
Intravasation
Metastasis (adherence to target organ endothelium)
Extravasation
Secondary Growth.
Oncogene Addiction
Unlike normal cells, tumor cells become dependent on activated oncogenes to survive. So turning off GF’s will kill cell, not just stop growth like it would in normal tissues.
Apoptosis
Programmed cell death. Very clean: cells shrink and condense.
Assays for Apoptosis
Acridine orange, DNA ladder to show 200 BP pieces of cell fragmentation, Flow cytometry to show that cell has less than 2N, TUNEL Assay where tags bind to breaks in DS DNA.
Annexin V and Phosphatidylserine Flip
Phosphatidylserine is normally on the intracellular leaflet. During apoptosis, it appears on the outer leaflet so cell can be eaten by macrophages. Annexin V stains phosphatidylserine.
Caspase Activation
Cleaved after specific aspartic acids, have a cysteine in the active site. Have large and small subunit. Initiator caspase (8/9) activates many executioner caspases (2,3,6,7).
Executioner Caspase-Mediated Cleavage
Executioner casperase (2,3,6,7) can cleave ICAD off of CAD (which is a nuclease), to degrade DNA into 200 BP slices.
Extrinsic Apoptosis Pathway
1) Ligand induced receptor trimerization (Fas ligand and Fas receptor, TNF, TRAIL)
2) Recruitment of receptor associated proteins (DISC).
3) Activation of initiator caspase 8, which will activate executioner caspases.
Intrinsic Apoptosis Pathway
Begins with loss of mitochondrial transmembrane potential. Cytochrome C is released from the mitochondrial membrane, which activates APAF1 to hydrolyze dATP to dADP. Activates the apoptosome, which activates procaspase 9. Caspase 9 activates executioner caspases.
BCL-2 Family of Proteins
BCl2 has all BH domains (4, 3, 1, 2) and is anti-apoptotic.
Bax and Bak are pro-apoptotic (don’t have BH4)
PUMA, NOXA, Bid are pro-apoptotic and only have BH3.
Bax and Bak
BCL2 family of proteins, are pro-apoptotic and have domains 3, 1, and 2. They form pores in the mitochondrial membrane to release cytochrome C, thus activating APAF 1, hydrolysis of dATP to dADP, apoptosome activation, casp-9 activation, and executioner casp activation.
Bcl2
Anti-apoptotic because it binds to bax and bak and prevents their activity.
Bid
BLH-3 only protein that directly activates Bax and Bak.
PUMA and NOXA
Upregulated by P53, blocks Bcl2 activation of Bax and Bak, so is pro-apoptotic.
Determination vs Differentiation
Determination is where a cell becomes committed to a single lineage. Differentiation is where a cell acquires specific properties that makes it a unique cell type.
Events associated with myoblast differentiation:
Exit from cell cycle, expression of muscle-specific genes, fuse to form multinucleated myotubes, express acetylcholine receptors.
MRF’s
Muscle regulatory factors are master regulators (transcription factors) that are necessary and sufficient for the lineage. These include MyoD, Myf5, myogenin, MRF4. They can each reprogram fibroblasts to the muscle lineage and play roles in determination and differentiation. Also work with transcription factors that drive muscle development.
MRF’s that control determination
MyoD, Myf5 can take a somite and turn it into a myoblast
MRF differentiation factors
Myogenin and Myf4 can differentiate a myoblast to a myofiber.
Muscle Creatine Kinase Gene Enhancer
Has binding sites for MRFs and MEF2s. Binding of these things drives expression of muscle creatine kinase and other muscle related proteins
Transcriptional feedback of MRF and MEF2
They regulate their own and each other’s expression to ensure proper amplification of the differentiation process.
How do MRF’s affect cell cycle regulators?
MRF induces the expression of p21, which causes cyclin D levels to drop and the cell cycle to arrest.
How are MyoD and Cell Cycle Machinery Linked?
Decreased growth factors decreases Cyclin D activity, which decreases phosphorylation of Rb. This causes cell cycle exit. Simultaneously, decreased growth factors increases MyoD transcriptional activity, which upregulates P21 expression, which decreases Cyclin E activity, and causes reduced pRb.
Difference between pluripotent and totipotent stem cells?
Totipotent can generate all cells, even extraembryonic ones. Pluripotent can generate all cells of an embryo.
SCNT
Somatic Cell Nuclear Transfer, where a somatic nucleus is transferred into an enucleated oocyte.
Therapeutic cloning
Where SCNT is used to generate ES cells, which are induced to differentiate for therapy.
iPS cells
Induced pluripotent cells. Where somatic cells are reprogrammed with Yamanaka cocktail (Oct4, Sox2, Nanog, c-myc).
