Cell Cycle, Apoptosis and Cancer Flashcards
What is cell cycle?
Process of cell growth, chromosome replication and division
What is the product of cell cycle?
2 daughter cells with the same genetic information
What if cell cycle becomes aberrant?
Unchecked cell growth/cancer
What are the stages of interphase, cell growth?
G1, S and G2
G1 phase (Gap 1)
RNA and protein synthesis due to mitogens and exogenous GFs. Proteins needed to replicate DNA in next phase.
S phase (synthesis)
DNA is replicated
G2 phase (Gap 2)
RNA and protein synthesis, integrity of DNA checked before mitosis
Mitosis
- Cell division, nuclear and cytoplasmic
- Creates 2 identical daughter cells
- Prophase, metaphase, anaphase, telophase and cytokinesis (PMAT)
Go phase (resting)
- Go cells have exited the cell cycle and are not growing or dividing
- They make just enough RNA + proteins for general housekeeping or specialized functions
- May re-enter cell cycle at G1 after stimulation by GFs
- Terminally differentiated cells can reenter by suppression of CKI, overproduction of cyclin D1, viral oncogenes
Restriction Point
- GFs are limiting –> cell cycle arrested in G1 at about 2h before starting S phase (restriction point R)
- When cells pass R pt, they are GF-independent and complete cell cycle
Checkpoints of Cell Cycle
G1, G2 and Metaphase
G1 Checkpoint
Verifies integrity of DNA, any DNA damage induces molecular mechanisms to arrest cycle at this point (mitogen deprivation)
G2 Checkpoint
Verifies completeness of genomic DNA replication before mitosis
Metaphase Checkpoint
Monitors attachment of chromosomes to the mitotic spindle before anaphase and chromosome segregation
DNA Content
- Resting cells in G1 have DNA content of 2N
- In S phase are between 2N and 4N
- 4N in G2
- 2N after cytokinesis
Labeling cells w/ DNA binding fluorescent dye
Able to sort them with FACS cells and different phases can be distinguished
3 cell types in the human body
- Permanent: remain in Go phase, can’t be regenerated (cardiac, neurons, RBCs)
- Stable/quiescent: ability to exit Go and enter G1 with GFs, regenerate damaged tissue (hepatocytes, kidney epithelial cells)
- Labile: Never enter Go, constantly dividing to replace lost cell population (gut, epithelium, skin, hair follicles)
Cyclin vs. Cyclin Dependent Kinases
Cyclin: Regulatory subunit
CDK: Catalytic subunit
Cyclin, CDK mechanism
Cyclin –> CDK –> partial activation of kinase activity –> direct complex to be phosphorylated by proteins
Full activation of CDKs require CDK-activating kinase (CAK)
Cyclin factoids
- There are many cyclins and CDKs in euk cells, each cyclin can bind to more than 1 CDK and vice versa
- Levels of CDKs are constant during cell cycle
- Levels of individual cyclins vary during the phases
Cyclin-CDK Complex During Cell Cycle
- Activity of CDKs varies in different phases of the cell cycle due to transient and cyclical availability of cyclins
- At start of G1, cyclin D complexes w/ CDK4 and 6, after cells pass through R pt and enter S phase, cyclin D is degraded
Cyclin-CDK Complex Stages During Cell Cycle
- cyclin E-CDK2 is active in G1–>S transition
- cyclin A-CDK2 is active in S phase to induce enzymes for DNA synthesis
- cyclin A-CDK1 and cyclin B-CDK1 initiate mitosis
Inhibitors of cyclin-CDK complex
WEE1 kinase, CKIs (CIP/KIP and INK4)
INK4 is specific to G1 CDKs
CIP/KIP bind to G1/S CDKs
Retinoblastoma protein
- RB = substrate of G1 and G1/S Cyclin-CDK complexes
- RB = tumor-suppressor protein b/c it can arrest the cell cycle at G1 checkpoint
- Hypophosphorylated RB binds to E2F –> Sequesters E2F and prevents from triggering transcription of cyclin E and cyclin A, DNA replication proteins
- Hyperphosphorylation of RB by G1, G1/S CDKs release E2Fs –> transcription of cyclin E so cells can transit G1 and enter S phase
- S phase and M phase cyclin-CDK complexes keep RB phosphorylated, degradation of these cyclins –> dephosphorylation of RB
p53 tumor suppressor gene
Re-write.
Proteolysis of Cyclins
Accomplished by polyubiquitination catalyzed by Ub-ligase. Specific Ub-ligase can ub CKIs to degrade them, releasing inhibition of S phase cyclin-CDK complexes
Failure of Checkpoints in Cell Cycle
- Result in cancers.
- DNA Damage Checkpoints (G1, G2)
- Spindle Assembly Checkpoint (M)
Activation of Cell Cycle
- Myc activation (oncogene)
- Active G1 CDK
- Inhibit Rb (phosphorylate)
- Release E2F
Hypo and hyperphosphorylation of RB
- Hypophosphorylated (active), RB blocks G1/S transition
- Hyperphosphorylation (inactive), RB allows G1/S transition
- E2F turns on Cyclin E and Cyclin A –> Activate CDK2 and keep RB inactive/E2F active
CDK needs 2 steps to be active
- Association of a cyclin
2. High levels of cyclin
Cyclin-CDK Complex
- Heart of cell-cycle control
- Causes changes in phosphorylation of substrates that regulate cell cycle events
- Cyclins are proteins that regulate CDKs, withouth it CDK is inactive
CDK Inhibitors
CKI’s –> CIP/KIP, p27
Inactivates kinase activity of CDK
Cyclin-CDK-CAK Mechanism
- Cyclin binds to CDK and causes T-loop to move out of active site
- CAK phosphorylates CDK at active site and activates enzyme
Four Major Classes of Cyclins in Vertebrate Cells
- Cyclin D (G1) – Complex with CDK4 and CDK6
- Cyclin E (G1/S) – Complex with CDK2
- Cyclin A (S) – Complex with CDK2
- Cyclins A/B (M) – Complex with CDK1
Regulation of Cyclin-CDK Activity
- Phosphorylation of CDK
- Binding of CKI P21
- Proteolysis of cyclins
Wee1 / CDC 25 CDK inhibitor/regulator mechanism
- Wee1 phosphorylates the “roof” site, inhibits CDK activity
- CDC25 is a phosphatase taht dephosphorylates “roof” site to increase CDK activity
CAK
CDK Activating Kinase
CKI Mechanism
- Binds to both CDK and Cyclin to inactivate
2. Used for control of G1/S CDKs and S CDKs
Anaphase-promoting complex or cyclosome (APC/C)
- Used to regulate protein degradation to progress mitosis from metaphse to anaphase
- APC/C is a member of ubiquitin ligase family of enzymes
- Eliminates cyclin S and M
APC/C activated by binding of CDC20
- Leads to polyubiquitinization of M-cyclin in M-CDK complex
- Because cyclins destroyed, most CDKs inactivated and dephosphorylated
Condition to move into anaphase
- Must get rid of S-cyclin and M-cyclin
p53 and tumor suppression
- p53 is a tumor suppression gene
- Tumors inactivate p53
- Allow further DNA damage to happen, cancer progression
DNA damage activates protein kinases
- Protein kinases phosphorylate and stabilize p53 protein
2. STOP CELL CYCLE
p53 activation/inactivation
- Phosphorylated p53 (active) increases transcription of p21, a CKI
- p21 binds and inactivates cyclin-CDK complexes, causing cell cycle arrest
- MDM2 keeps p53 inactive
Apoptosis Pathways (2)
- Intrinsic
- Extrinsic
Goal is to maintain cell integrity
Intrinsic apoptosis defining characteristics
- Depend on internal stimuli, abnormalities in DNA
- Mitochondrial dependent (mito dysfunction)
- BAX/BCL-2 are key regulators
Extrinsic apoptosis defining characteristics
- Depend on external stimuli, removal of survival factors, proteins of TNF family
- Mitochondrial independent
Common to both apoptosis pathways
Caspase 3 is activated in both pathways
Activation of caspases is key to apoptosis
Precursor to caspase
Procaspase, activated by protease cleavage
Form large/small subunit (tetris piece)
2 major classes of caspases
- Initiator caspases: initiate apoptosis, caspase 8/9
2. Executioner caspases: destroy targets, executes apoptosis (caspase 3)
Extrinsic apoptosis pathway
- Extracellular signals bind to CELL SURFACE DEATH RECEPTORS
- Death receptors have 3 classic domains
Extrinsic apoptosis mechanism
- Fas ligand binds to Fas Death receptor
- Recruits FADD adaptor protein
- Activates Caspase 8/10 –> Caspase 3 (executioner)
Intrinsic apoptosis mechanism
- Cytochrome C is released from mito, binds to pro-caspase activating adaptor protein APAF1
- Apaf1 forms APOPTOSOME which activates caspase-9
- Caspase-9 activates executioner caspase-3
BAX (BH123) protein in apoptosis
- BAX proteins become activated and aggregate in outer mito membrane, induce release of cytochrome C
- Apoptosome formed by binding to APAF1
BCL-2
Anti-apoptotic BCL proteins
- Prevent apoptosis by binding to pro-apoptotic proteins BAX/BH123 to prevent aggregation into active form
Intrinsic pathway summary
BAX induces cytochrome C release –> APAF1 complex –> Apoptosome –> Caspase 9 –> Caspase 3 (executioner caspase) –> Apoptosis
Slide 42 REVIEW PLEASE
Apoptosis vs. proliferation
2 hit hypothesis of cancer
Most of the time need 2 “hits” in order to get cancer – hereditary/sporadic
Retinoblastoma – RB Protein
Major regulator of cell cycle/apoptosis
Retinoblastoma facts
- Rare human cancer (1:20000)
- Occurs in childhood
- Result of cell cycle defects
Hereditary vs. Sporadic Retinoblastoma
Hereditary form inherits LOF/deletion of one copy of RB in every cell. Need to lose that copy to form tumor. Get tumor in both eyes.
Sporadic form needs both copies to have mutation. One eye tumor.
Somatic event causes mutations.
Proto-oncogene vs. oncogene
- Proto-oncogene encode proteins that promote cell growth and division
- Converted to oncogenes through gain of function mutations
- Oncogenes cause cell proliferation, express oncoproteins
Ras/Src are oncogenes
Common Oncogenes
- NEU -> RTK activation w/o ligand –> Breast cancer
- ERBB –> RTK always on –> Glioblastoma
- BCR/ABL fusion protein –> Chronic myelogenous leukemia
Review Slide 49
Gain of Function Mutations in Proto-oncogenes
Gain of function mutations and Loss of function mutations
GOF for Proto-oncogenes and LOF for Tumor supressor genes
Loss of APC is associated with cancer
Results in overexpression and cell proliferation
Loss of tumor-suppressor gene
Increase in cell surface signaling receptors associated with cell proliferation
Early adenoma
Best stage for early detection of cancers
Hanahan and Weinberg Hallmarks of Cancer Cells
- Self-sufficiency in growth signals
- Evade growth suppressors
- Activate invasion and metastasis
- Enable replicative immortality
- Inducible angiogenesis
- Resist cell death
- Deregulate cellular energetics
- Avoiding immune destruction
- Tumor-promoting inflammation
- Genome instability and mutation
Viral Oncogenesis
- Virus integrates proto-oncogene to host
- Proto-oncogene converted to oncogene by mutation
- Mutation occurs during viral replication (very inaccurate, no DNA repair)
- c-Src (normal proto-oncogene) vs. v-Src (oncogenic)
