Cancer Pathways, Apoptosis, and Cell Differentiation

Question 1

P16 Pathway

Answer 1

P16 inhibits Cyclin D and CDK4/6, that inhibition will inhibit the phosphorylation of Rb, which will inhibit E2F.

Question 2

E2F activation

Answer 2

Leads to proliferation, but also to activation of ARF, which inhibits MDM2, leading to the upregulation of p53. This is a feedback mechanism.

Question 3

Loss of APC

Answer 3

Causes Beta catenin to be available in the cell, increasing cyclin D and myc.

Question 4

Myc

Answer 4

Increases the activity of ARF, but also promotes cell proliferation

Question 5

PI3K Pathway

Answer 5

PI3K turns PIP2 into PIP3. PIP3 activates AKT, which promotes cell survival. PTEN inhibits PI3K.

Question 6

AKT

Answer 6

Promotes cell survival, activated by PIP3

Question 7

PTEN

Answer 7

Inhibits PI3K, which prevents PIP3-induced activation of AKT. Thus, PTEN causes apoptosis.
In cancer, PTEN can be mutated causes increased tumor survival

Question 8

Multi-step model of tumorigenesis

Answer 8

Normal epithelium, (loss of APC) hyperplastic epithelium, early adenoma, intermediate adenoma, late adenoma, (loss of p53) carcinoma, invasion and metastasis.

Question 9

CDKN2A Locus

Answer 9

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.

Question 10

Transition from epithelial cells to cancer cells

Answer 10

Cell-cell junctions lost (cadherins), loss of polarity, migration increased.

Question 11

Steps of metastasis

Answer 11

Angiogenesis (FGF and VEGF)
Invasion - Tumor cells attach to ecm, proteases degrade matrix.
Intravasation
Metastasis (adherence to target organ endothelium)
Extravasation
Secondary Growth.

Question 12

Oncogene Addiction

Answer 12

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.

Question 13

Apoptosis

Answer 13

Programmed cell death. Very clean: cells shrink and condense.

Question 14

Assays for Apoptosis

Answer 14

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.

Question 15

Annexin V and Phosphatidylserine Flip

Answer 15

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.

Question 16

Caspase Activation

Answer 16

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).

Question 17

Executioner Caspase-Mediated Cleavage

Answer 17

Executioner casperase (2,3,6,7) can cleave ICAD off of CAD (which is a nuclease), to degrade DNA into 200 BP slices.

Question 18

Extrinsic Apoptosis Pathway

Answer 18

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.

Question 19

Intrinsic Apoptosis Pathway

Answer 19

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.

Question 20

BCL-2 Family of Proteins

Answer 20

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.

Question 21

Bax and Bak

Answer 21

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.

Question 22

Bcl2

Answer 22

Anti-apoptotic because it binds to bax and bak and prevents their activity.

Question 23

Bid

Answer 23

BLH-3 only protein that directly activates Bax and Bak.

Question 24

PUMA and NOXA

Answer 24

Upregulated by P53, blocks Bcl2 activation of Bax and Bak, so is pro-apoptotic.

Question 24

Determination vs Differentiation

Answer 24

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.

Question 25

Events associated with myoblast differentiation:

Answer 25

Exit from cell cycle, expression of muscle-specific genes, fuse to form multinucleated myotubes, express acetylcholine receptors.

Question 26

MRF’s

Answer 26

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.

Question 27

MRF’s that control determination

Answer 27

MyoD, Myf5 can take a somite and turn it into a myoblast

Question 28

MRF differentiation factors

Answer 28

Myogenin and Myf4 can differentiate a myoblast to a myofiber.

Question 29

Muscle Creatine Kinase Gene Enhancer

Answer 29

Has binding sites for MRFs and MEF2s. Binding of these things drives expression of muscle creatine kinase and other muscle related proteins

Question 30

Transcriptional feedback of MRF and MEF2

Answer 30

They regulate their own and each other’s expression to ensure proper amplification of the differentiation process.

Question 31

How do MRF’s affect cell cycle regulators?

Answer 31

MRF induces the expression of p21, which causes cyclin D levels to drop and the cell cycle to arrest.

Question 32

How are MyoD and Cell Cycle Machinery Linked?

Answer 32

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.

Question 33

Difference between pluripotent and totipotent stem cells?

Answer 33

Totipotent can generate all cells, even extraembryonic ones. Pluripotent can generate all cells of an embryo.

Question 34

SCNT

Answer 34

Somatic Cell Nuclear Transfer, where a somatic nucleus is transferred into an enucleated oocyte.

Question 35

Therapeutic cloning

Answer 35

Where SCNT is used to generate ES cells, which are induced to differentiate for therapy.

Question 36

iPS cells

Answer 36

Induced pluripotent cells. Where somatic cells are reprogrammed with Yamanaka cocktail (Oct4, Sox2, Nanog, c-myc).