Exam 2 Flashcards
(91 cards)
1
Q
R point
A
The point after which cells are committed to go through the cell cycle
2
Q
Which cell cycle has risen R point in?
A
G1
3
Q
Name all stages of mammalian cell cycle
A
G0: quiescent stage
G1: decision making step with respect to outside signals
S: DNA replication
G2: make sure DNA replication was done correctly
M: mitosis
4
Q
Define CDK
A
Cyclin dependent kinase
-serine/threonine kinases that depend on cycling for activation of kinase activity and access to substrates
5
Q
Which cycling are active in which cell cycle phase?
A
Cycling A- increases in concert with entrance to S
Cyclin E- increases after R point and drops in S
Cyclin B- only active during M
Cycling D- active in G1 before R point and responsive to extracellular motigens
6
Q
What does a CDK inhibitor do? How many are there? Name them.
A
Negatively regulate cyclin-CDK complexes
- 7
- p15 p16 p18 p19 p57 p27 p21
7
Q
What can stimulate CKIs? (2)
A
- TGF-B
2. P21
8
Q
How does TGF-B stimulate CKI?
A
TGF-B is a tumor suppressor that increases p15 to inhibit cell growth before R point
9
Q
How does p21 stimulate CKI?
A
P21 senses DNA damage signals and inhibits various cyclins
10
Q
What can inhibit CKI expression?
A
Motigenic signals
-inhibitors can be sequestered in cytosine so that they can’t go to nucleus to bind to transcription factor
11
Q
Define Rb
A
Rb is a large nuclear phospho-protein with a lot of serine and threonine residues, which are good sites for phosphorylation
12
Q
What happens when Rb gets phosphorylated?
A
Its conformation changes
13
Q
What regulates Rb phosphorylation?
A
Cell cycle
14
Q
Describe Rb phosphorylation states at each phase: G0, G1 before R, R, G1 after R, S-G2-M
A
G0 - unphosphorylated G1 before R - hypophosphorylated R -hyperphosphorylated G1 after R- hyperphosphorylated S-G2-M- hyperphosphorylated
15
Q
Models of RB fxn in cell cycle by studying viral oncoproteins
HOW DO WE KNOW RB IS AN IMPORTANT TARGET TO INACTIVATE DURING VIRAL TRANSFORMATION?
A
Found three unrelated diseases with Rb target binding
16
Q
Models of RB fxn in cell cycle by studying viral oncoproteins
HOW DO WE KNOW THAT HYPO-PHOS RB STATE MUST BE THE ONE CAPABLE OF INHIBITING PROLIFERATION?
A
Viral oncoproteins could only bind to hypo-phosphorylated state of Rb
17
Q
Models of RB fxn in cell cycle by studying viral oncoproteins. What did it tell us?
A
DNA tumor viruses induce transformation of cells through inactivating the Rb tumor suppressor protein
18
Q
How does Rb guard the R point?
A
Rb controls cell cycle progression at early to mid G1.
Rb is inactivated by hyper phosphorylation once the cell passes R point.
Rb, through its phosphorylation, serves as the guardian of the R point.
19
Q
What determines Rb phosphorylation?
A
Cyclin-CDK COMPLEXES
1st: D-CDK4/6 changes from unphosphorylated to hypophosphorylated
2nd: E-CDK2 changes from hypophosphorylated to hyperphosphorylated
20
Q
What happens to Rb’s tumor suppressor functions when it’s hyper-phosphorylated?
A
Lost.
Some tumors inactivate Rb by keeping it in hyperphosphorylated state
21
Q
What does active Rb do? What are its active states?
A
Rb is active when it is un- or hypo- phosphorylated.
Active Rb has pocket formation to bind E2F and prevent transcription.
22
Q
What happens once Rb binds E2F? (2)
A
- Prevents E2F from binding its transcription co-activators (HAT)
- Recruits transcription co-depressor (HDAC)
23
Q
What happens when Rb in inactive hyper-phosphorylated state and can’t bind E2F?
A
E2F COMPLEX IS IN ITS ACTIVE FORM TO ACTIVE TRANSCRIPTION.
- Binds to sequence to enable entry into S phase
- Recruits transcription co-activator (HAT)
- Binds DP1 and 2 subunits to form heterodimeric complexes that then bind promoters
- Activate cyclin E genes and genes involved in DNA REPLICATION
24
Q
What is E2F?
A
Sequence specific transcription factor that enables entry into S phase
25
Positive feedback regulation of E2F:
E2F increases expression of cyclin E, which then activates more CDK2 to induce hyperphosphorylation of more Rb, leading to more activation of E2F
26
Negative feedback regulation of E2F:
Inactivation if E2F in S phase by cyclin A-CDK2. As cells enter S phase, cyclin A-CDK2 complex phosphorylated the E2F and DP subunits, resulting in dissociation of the subunits and loss of transcription activity.
27
Review of Rb and cell cycle pathway: start from extracellular signals and end at DNA replication
Extracellular signals --D-CDK4/6 - - Rb hypophosphorylated in G1 -- + E-CDK2 -- Rb hyperphosphorylated at R point -- E2F active -- entry into S phase and replication
28
Different cell cycle regulators in human cancer (5)
1. Tyrosine receptor kinase pathway
2. R point
3. Myc
4. P16 and melanoma
5. TGF-B pathway
29
RTK MITOGENIC SIGNALING PATHWAY: start with GFs and end with phosph of Rb
GFs- RTKs- Ras - AP1 (Jun/Fos) - bind to cyclin D1 promoter and activate its transcription - phosphorylation of Rb to inactivate it - active E2F - S phase entry
30
RTK MITOGENIC SIGNALING PATHWAY: how was pathway elucidated?
Dominant negative mutants-
1. DN Ras -- no growth no matter how much GF you add
2. DN Ras and KO Rb -- cell growth, so Rb is downstream if Ras
31
Cell cycle regulators: R point : what is up regulated to promote cell cycle in cancers? (4)
Cyclin D
CDK 4/6
Cyclin E
CDK2
32
Cell cycle regulators: R point : what is downregulated to promote cell cycle in cancers?
Rb
P16
P27
Down regulating these prevent them from inhibiting the cell cycle
33
Cell cycle regulators - Myc. What is Myc?
Myc is a nuclear transcription factor that binds sequence specifically to E box, and when deregulated is an oncogene to promote cell growth
34
Myc family: (4)
Myc
Max
Mad
Miz
Form homo or heterodimers to activate or repress transcription depending on combo of complex
35
What does Myc-Mx do? What are its target genes?
Activates transcription.
MITOGENIC signals -- increased Myc expression -- Myc-max activates transcription of target genes
Target genes: cyclin D, CDK4, E2F
36
What does Mad-Max do? What are its target genes?
Represses transcription.
Differentiation signals -- high mad levels kick of Myc -- Mad-Max represses transcription of genes involved in cell growth
37
Myc has multiple mechanisms to coordinate activity in cancer (2)
1. Myc-Max increases expression of genes that promote growth
2. Myc-Miz decreases expression of genes that can block cell cycle
38
Cell cycle regulators: P16 and melanoma -- what is familial melanoma (3 grps)
Malignant and needs surgical removal
3 grps:
1. Sporadic melanomas that cluster in families
2. Familial melanomas that result from low risk alleles
3. Hereditary melanoma because of high risk, highly penetrant alleles
Linkage analysis mapped responsible gene to p16 tumor suppressor gene
39
Cell cycle regulators: P16 and melanoma -- what is p16?
P16 is a CKI WHICH MAKES IT A TUMOR SUPPRESSOR
40
Cell cycle regulators: P16 and melanoma : normal function of P16?
Inhibits cyclin D/CDK4 to block phosphorylation of Rb so that cell can't pass R point of cell cycle
41
Cell cycle regulators: P16 and melanoma : what does her line mutation of p16 do?
- impairs ability of p16 to bind to and inhibit CDK4
| - Germaine mutation of p16 is a predisposition for melanoma
42
Cell cycle regulators: P16 and melanoma - other causes of melanoma (2)
- sporadic : p16 is silenced by promoter methylation
- sporadic and familial cases : mutation is CDK4 prevents it from binding to CKIs, so it still binds cyclin-D and phosphorylated Rb
*CDK4 is a dominant negative allele : only needs one copy to escape inhibition by p16
43
Cell cycle regulators : TGF-B - what is it?
Transforming growth factor B that inhibits cell growth
It's a small molecule/ligand that binds to cell surface receptor
44
Cell cycle regulators : TGF-B -- what are its two activities depending on context?
In context of normal tissue, benign tumor, and early stages of malignancy-- TGF-B is a tumor suppressor because it stop s proliferation
THEN
Inactivation of growth inhibitory factor
THEN
in context of invasive metastatic tumor -- TGF-B acts as a tumor promoter
45
Cell cycle regulators : TGF-B -- pathway!
1. TFG/B binds to cell surface receptor by bringing together Type I receptor (usually inactive unless bound by ligand) and Type II receptor (constitutive my active)
2. Active kindase on receptor phosphorylated Smad proteins -- Smad 2/3
3. Once phosphorylated, Smad2/3 forms complex with Smad 4
4. Smad 3/4 complex targets DNA seq and forms complex with Miz-1
5. Major effect: increased production of P15, which binds cyclin D-CDK4/6 complex and leads to cell cycle arrest
6. Minor effect: on cyclin E -CDK2 complex -- leads to cell cycle arrest
46
Cell cycle regulators : TGF-B: inactivation of TGF-B pathway in human cancer (2)
1. Mutations in smad2 leads to colorectal cancer
| 2. Mutations that inactivate smad4 lead to Pancreatic cancer
47
POSITIVE REGULATORS OF CELL CYCLE PROGRESSION
Oncogenes
RTK pathways
Myc oncogenes
48
NEGATIVE REGULATORS OF CELL CYCLE PROGRESSION
Tumor suppressor genes
TGF-B pathway
Familial melanoma and p16
49
Most frequently mutated gene in human cancers
P53
| Ovary, esophagus, colorectum, etc
50
P53-- Li-Fraumeni Syndrome
People with this disease are susceptible to a wide array of cancers
- susceptibility gene mapped to p53 protein (17p13)
- 70% of families with syndrome showed mutant p53 alleles transmit in Mendelian fashion
- age of onset is variable
51
P53 discovery -- what technique and what kinds of cells did they use?
Immuno precipitation and SV40 large T transformed cells
52
What is immuno precipitatio?
Using antibodies to isolate prep town of interest and its associated proteins
53
Describe immuno precipitation process to discover p53
1 SV40 large T transformed cells were labeled with S-met (initiation codon, so not all large T proteins and associated proteins are labeled)
2 Lyse labeled cells with SDS detergent to break lipid bilayer
3 incubate cell extract (protein solution mix) with anti-large T antibody conjugated to tagged beads
4 large T protein will bind to ab, as will other proteins associated with large T protein
5 precipitate the beads, then boil in SDS Sample buffer to denature the proteins and separate from the beads
6 run the separated proteins on acrylamide gel and visualize by S-Met labels
Result: p53 identified as a SV40 large T antigen associated protein with molecular mass of 53 kDa
54
Initial finding of p53 role in transformation : describe it
1. Extract p53 band from immunoprecipitation experiment and generate antibodies from it
2. Clone p53 cDNA from cancer cell line
3. Take normal rat fibroblasts that have Ras oncogene introduced
4. Add p53 in addition to Ras oncogene
5. See transformed cells
Cancer cells had p53 but normal cells didn't
People though p53 was an oncogene
55
Initial finding of p53 role in transformation : why did they add Ras oncogene to the rat fibroblasts?
Ras oncogene alone won't cause transformation so it'll help us see whether adding p53 will cause transformation
56
Describe the later experiment that showed p53 is a tumor suppressor
1. Adding WT P53 from normal cells and Ras oncogene to cells lead to NON-TRANSFORMED CELLS
2. Adding mutant p53 from Tumor cells and Ras oncogene to cells led to TRANSFORMED CELLS
Conclusion: WT P53 suppresses the transforming activity of the Ras oncogene
-mutant p53 acts in dominant fashion
57
Why does p53 inactivation not follow Knudson's model of 2-hit elimination process?
Majority of tumor-associated p53 mutation are mis sense mutations (introduce point mutation) rather than nonsense mutations (STOP)
58
In which two ways is p53 behavior unlike that of other tumor suppressors?
1. Inactivation of p53 doesn't follow the Knudson's 2-hit elimination process
2. Majority of tumor-associated p53 mutation are mis sense mutations (introduce point mutation) rather than nonsense mutations (STOP)
59
Regulation of p53 (2)
1. Dominant negative allele
| 2. P53 forms homotetramers
60
What is a dominant negative allele?
Mutant acts in dominant manner to inhibit function of the WT allele when expressed in the same cell
61
How does forming homotetramers regulate p53?
If any one subunit is wrong, then the whole unit doesn't work.
With equal amount of WT and mutant P53 subunits, only 1/16 of the p53 complexes will contain all WT p53 subunits and be functional.
62
Two ways to inactive p53
1. Mis sense mutations act as dominant negative alleles-- missense mutations are more efficient than null mutations because it can inactivate 15/16 of p53 rather than leave 1/2 of o53 in WT form
2. Inactivate the WT allele by LOH -- to get rid of the leftover 1/16 of functional p53
63
What is p53?
P53 is a transcription factor, a nucleoprotein that binds to DNA
P53 can bind to promoter DNA with consensus sequence repeated 2x in tandem -- promoter sequence is loose, so p53 can actually bind and activate expression of many genes
64
What are p53 target genes involved in?
```
Cell cycle arrest
Apoptosis
P53 antagonist
DNA repair
Anti-angiogenesis
```
65
phenotypes of apoptosis
overall shrinkage of cell volume and nucleus (chromatin compaction); loss of adhesion to neighboring cells; blebbing; DNA fragmentation; phagocytosis of dying cell
66
INTERNAL STIMULI OF APOPTOSIS
abnormalities in DNA
67
EXTERNAL STIMULI OF APOPTOSIS
removal of growth factors; addition of cytokines (TNF-a)
68
extrinsic pathway overview
ligands --> death receptor trimerization --> FADD/DISC --> caspase 8, 10 --> caspase 3 --> cleave targets
69
intrinsic pathway overview
intrinsic apoptosis signals --> Bax dimers --> cytochrome C translocation --> cyt C binds to Apaf1 in cytosol to form apoptosome --> caspace 9 -> caspase 3 --> cleave targets
70
caspase stands for:
cysteine aspartyl specific protease -- cysteine active site cleaves proteins with Asp residue
71
Bcl-2 is an oncogene or tsg? why?
oncogene; promotes survival of lymphoid cells by blocking apoptosis (B cell Leukemia when bcl-2 gene translocated to be under IgH chain enhancer)
72
how do Bcl-2 family proteins homo- and hetero- dimerize?
through shared BH3 domains
73
cellular targets of caspases (5)
1. FAK -- focal adhesion kinase
2. Lamins
3. proteins required for cell structure
4. endonuclease CAD
5. enzymes involved in DNA repair
74
How does TUNEL assay measure apoptosis?
measures DNA fragmentation
- TdT adds dNTPs to the 3' end of DNA molecules in absence of template, so when cells undergo apoptosis, there will be many broken chromosomes with free 3' ends
- attach a FITC probe to added nucleotides to visualize
75
Three ways p53 promotes apoptosis
1. activate expression of Fas receptor
2. activate expression of Bax and other pro-apoptotic proteins
3. activate expression of IGF-BP3, which inhibits IGF-1 activity
76
Two ways p53 promotes cell cycle arrest
1. activates expression of p21c1p1 --> p21 inhibits CDK1 and CDK2
2. activates expression of 14-3-3sigma --> 14-3-3sigma binds to cyclinB-CDK1 to inhibit activity
77
p53 regulates which two cell cycle checkpoints?
G1 and G2
78
current model of p53: p53 can detect DNA damage and ___
1. trigger cell cycle arrest to give cells time to repair the damage
2. send cells that can't repair damages to die in apopptosis
79
why is p53 an unstable protein?
high degradation rate
80
what kinds of cellular stress activates p53?
- lack of nucleotides
- UV radiation
- ionizing radiation
- oncogene signaling
- hypoxia
- blockage of transcription
81
what happens with too much p53?
premature aging -- mice with superactive p53 get less cancer but age quicker b/c they're now really sensitive to any cellular stress, so a lot of tissues undergo apoptosis --> shorter lifespan
82
is Mdm2 an ocogene or tsg?
oncogene -- it is anti-apoptotic because it binds p53 and inhibits its transcription activity and directs ubiquitination of p53
83
DNA damage --> ATM/ATR kinases --> (2)
1. kinases phosphoryalte p53 --> Mdm2 can't bind phosphorylated p53, so p53 is stabilized for transcriptoin
2. kinases phosphorylate Mdm2--> phosphorylated Mdm2 can't ubiqunitinate p53, so p53 is stabilized for transcription
84
What negatively regulates Mdm2?
p14ARF
85
what activates Mdm2?
mitgentic signaling
86
what can activate p14ARF?
excessive amounts of E2F, causing p14ARF to bind and sequester Mdm2 in nucleolus, thus blocking its access to p53
87
p14ARF monitors deregulation of Rb -- how?
deregulation of Rb -- excess E2F -- p14ARF activated -- p53 increase -- apoptosis!
88
which two critical tumor suppressors are derived from the same gene locus?
p14ARF and p16INK4A
- alternative splicing results in different reading frames
- P16INK4A --> blocks Rb phosphorylation --> blocks passage past R point
- p14ARF --> p53 --> cell cycle arrest, apoptosis
89
Role of Mdm2 in human cancer -- SNP at?
SNP at nt309 (promoter region of Mdm2)
90
role of Mdm2 in human cancer -- what happens?
results in increased binding of transcription activator to Mdm2 promoter, so elevated levels of Mdm2 protein --> reduced p53
-population with 4-fold elevated levels of Mdm2 protein had earlier onset of tumors b/c they had less p53
91
cancer derived mutations usually abolish which two activities of p53?
DNA binding and transcription activity
