Lectures 3-4 Flashcards
Types of inherited single gene defects
-Retinoblastoma, a childhood eye cancer (Rb)
-Li-Fraumeni syndrome (p53)
-BRCA1
-2 predisposition to breast and ovarian cancer (APC)
Two forms of Retinoblastoma
-Familial (Hereditary): Multiple tumors, affecting both eyes, early onset, 6x increased cancer risk.
-Sporadic (Non-hereditary): Single tumor, affecting one eye, later onset.
Phases of cell cycle:
-G1
-S
-G2
-M
-G0
G1:
Cell make decisions about growth versus quiescence
S phase:
DNA is synthesized
G2:
Making sure all the DNA is replicated with fidelity
M Phase:
-2 new cells are made through the division of the genetic material
-mitosis (prophase, prometaphase, metaphase, anaphase, telophase) and cytokinesis
G0:
Resting, nonproliferative state of cells that have withdrawn from the active cell cycle
What features do checkpoints have:
-Allow internal feedback control
-Allow control from external stimuli
Types of cell cycle checkpoints:
-G1/S (determines if DNA synthesis can begin)
-Intra-S and G2/M (Prevents the replication of damaged DNA until repaired)
-Spindle Checkpoint (ensures chromosomes are aligned correctly to spindle. If not mitotic arrest doesn’t proceed to Anaphase)
Progression to S phase depends on?
The presence of growth factors in the G1 phase up until the restriction point.
What is the restriction point.
A checkpoint where a cell must make the decision of whether to proceed to S or to enter G0. At this point growth factors no longer influence progression.
CDK Inhibitors:
-INK4/CDKN2 (p16, p15, p18, p19).
-CIP/KIP/CDKN1 (p21cip1, p27kip1)
How does INK4 inhibit?
They bind to CDK4/6 which prevents it from binding to cyclin D
How does the CIP/KIP/CDKN1 inhibit?
They can inhibit all of the other cyclin-CDK complexes by blocking their ATP binding site.
What is CAK
-Stand for CDK activating kinases.
-Work by phosphorylating the activation loops.
What is CD25
It is a class of phosphates that remove inhibitory phosphorylation’s that affect CDK2
How are cyclin Ds activated?
By growth factors causing a signaling cascade resulting in transcription factor AP-1 which regulates key cyclins.
How is cyclin E/CDK2 activated?
CDK4/6 sequestering p21 and p27
Function of active cyclin E-CDK2:
It phosphorylates p27 which marks it for degradation by the proteasome, allowing for more active cyclin E-CDK2 complexes to be generated and leading to hyperphosphorylation of RB.
What happens when RB is hyperphosphorylated
Hyperphosphorylation makes RB inactive which allows for the release of E2F and HDAC which are needed for S phase gene expression.
Nuclear RB protein is a member of?
The “pocket proteins”
What is HDAC
It is an enzyme (histone deacetylase) that when bound to RB it blocks S phase gene expression.
What is E2F
-E2F is a key transcriptional regulator of S-phase genes
-Positive regulator of cell cycle progression
-Active E2Fs1,2,3 induce expression of late G1 genes, required for S phase.
How is E2F1 degraded
In the G1/S transition cyclin A and CDK2 phosphorylates both E2F and DP subunits; leading to their dissociation, and loss of transcriptional activities.
What are E2Fs1,2 and 3a
They are Hsp (heat shock protein)-chaperones also know as ‘activating E2Fs’
S-Phase gene expression leads to:
-DNA polymerase, topoisomerase, ligase etc
-Initiation Factors
-Structural proteins (e.g. Histones)
-Proteins involved in synthesizing DNA precursor nucleotides
-Spindle assembly checkpoint protein e.g MAD2
What cyclin is involved in G2/M Phase Progression
Cyclin B partnered with CDK1
What keeps CDK1 inactive
The kinases WEE1 and MYT1
What activates CDK1
Two independent events:
1. CAK-mediated phosphorylation
2. Cdc25-mediated dephosphorylation
Targets of Active CDK1/Cyclin B:
– phosphorylates serine residues on lamin (breakdown of nuclear membrane)
– Phosphorylates histones
– Phosphorylates microtubule associated proteins (altered behavior during mitosis)
What are CDC25 Phosphatases
-They are a family of phosphatases (A, B, C) that dephosphorylate specific tyrosine and
threonine residues on CDKs (which inhibit CDK function)
-Also dephosphorylates inhibitory phosphorylation residues on CDK2 in G1
Which cell cycle stages are CDC25s involved in
G1-S and G2-M transitions.
Why are CDC25 phosphatases targets of the checkpoint machinery during DNA damage
Because they allow for progression through the cell cycle, so in order to prevent this they are inactivated or degraded.
Overexpression of CDC25A and B is seen in?
High grade tumors and poor prognosis cancers.
What are checkpoint kinases (CHK)
They are kinases activated in response to DNA damage and target CDC25.
What is Spindle Assembly Checkpoint
It ensures proper segregation of sister chromatids by inhibiting metaphase to anaphase transition until all chromosomes are attached correctly to the mitotic spindle.
What are Cohesins?
Protein complexes that hold together sister chromatids.
What is separase?
It is a protease which cleaves cohesins
What happens when chromosomes are unaligned?
Separase is kept inactive by securin and Cdk1-cyclinB
What happens when chromosomes become aligned?
CyclinB and securin are ubiquitinylated by the Anaphase Promoting Complex (APC)-CDC20 complex and degraded. This leads to the activation of separase, which releases sister chromatids, allowing for metaphase to anaphase transition.
MYC represses expression of what?
p15 and p21
TGFb reduces what expression?
- c-MYC expression-Smad3 and E2F4/5
- p107 shut down c-MYC expression
TGFb-mediated repression of MYC is lost in what cancer?
Breast cancer
How does TGFB induce expression of p21 and p15?
TGFb signalling: Smad2/3 associate with Smad4 heterotrimeric complex moves to nucleus and collaborates with the transcription factor Miz-1 to induce expression
P21 and P15 are repressed in what cancers?
Any cancers where MYC is overexpressed
How does TGFb block cell cycle progression.
By preventing phosphorylation of RB, which prevents it from releasing E2F.
What is Akt
It is a serine/threonine kinase which is activated downstream of growth factor receptors. Its function is to promote cell cycle progression.
3 functions of Akt:
-Inhibition of CDK Inhibitors (p21 and p27).
-Promotes cell cycle progression by blocking the actions of GSK3b.
-Blocks the actions of a pro-apoptotic protein Bad.
How does Akt inhibit CDKIs p21 and p27?
Akt phosphorylates them which leads to their translocation from the nucleus to the cytoplasm where they are degraded by the proteosome
Targets of GSK3b
It has inhibitory actions on MYC and CyclinD1.
What does Autonomous cell division mean?
The cell no longer depends on positive and negative growth factors (i.e cells are now cancerous).
What pathways play a central role in transmitting oncogenic signals.
-The PI3K-AKT pathway
-The RAS-RAF-MEK-ERK pathways
Increased Akt activity is frequent in what cancers?
Breast cancer.
Tumor grade can be linked with?
Higher pAKT expression and reduced nuclear P27.
