Cancer Bio - Exam 2 Flashcards
Most signaling molecules are what?
Proteins
Proteins on the plasma membrane that bind to a specific ligand and transmit signal into the cells
Receptors
What two types of molecules can pass the plasma membrane?
Small and hydrophilic
Animal cells depend on ____ to divide (without, they can not).
Growth factors
Most cancer is induced in what type of pathway?
Cancer pathway
Why does signaling matter?
Error in signaling can lead to cancer formation
Not all ____ can cause cancer, only certain molecules related to cancer can cause cancer to form
Mutations
ErbB signaling network: How do cells communicate with their surroundings?
The ligands will bind to the receptors (growth factor receptors) in the plasma membrane.
Release adaptors and enzymes, causing signaling cascades in cytosol.
Causes transcription factors to bind.
5 stages of ErbB signaling network
Apoptosis (cell death)
Migration
Growth
Adhesion
Differentiation
Normal metazoan cells control what?
Each other’s lives
In order to creat normal tissue structure and function, the different cell types must what?
Coexist
The relative numbers and positions of each cell type must be what?
Tightly controlled
The cellular control is largely achieved via what?
The exchange of signals between cells within a cell type, and between different cell types.
(Can communicate between tissues)
Control is achieved by?
Exchange of signals between cells within a cell type and between different cell types
All the decisions made by an individual cell about its proliferation must represent what type of decision?
A consensus decision shared with cells that reside in the neighborhood.
Think: cell division, migration, death
Provide stem cells for renewal of the epithelium; stem cells that will become differentiated into epithelial cells.
Crypt
As cells divide, they more differentiated cells will move in which direction?
Upward; at the highest point, they will be well-differentiated, and they can survive up to a few days
All cell interaction is coordinated and occurs through what?
Through signal transduction
Src is what molecule?
A tyrosine kinase and acts as a signal transduction protein
What evidence proves that a single protein can affect many different behaviors/aspects of a cell?
Src affects other proteins and many different cellular functions. Src is can signal to affect other aspects of cell behavior and functions (i.e. division, etc.)
Technique used to detect proteins in the cell (ex. used to detect Src).
Western blot
Antibody in Src experiment was used against:
2-P-Y (phosphorylated tyrosine)
Once some proteins become phosphorylated, they can function as
signaling molecules
3 types of kinases:
Threonine (T), serine (S), tyrosine (Y)
What make up 99.9% of kinases?
Serine kinase and threonine kinase
Tyrosine kinases make up what portion of kinases?
<0.1%, but known to play critical role in cell division
When testing for Src-transformed NIH 3T3 cells and you run gel what would you expect to see?
Proteins will be separated by size (smaller = faster and larger = slower)
The first protein that the growth factor binds to
EGF-R (epidermal growth factor receptor)
All tyrosine kinase receptors (RTKs) consists of what three functional domains?
(hint: think of EGF-R domains)
Ectodomain (extracellular domain): 621 AA
Transmembrane domain: 23 AA
Cytoplasmic domain: 542 AA
Part of the cytoplasmic domain shows what with Src?
Homology
Once you add EGF-R, what happens to the cell?
Once growth factor is added, cells immediately start to divide (cells controlled by growth factor)
Can growth factors pass through the plasma membrane to control cell division?
No, but control cell division extracellullarly by binding immediately to the surface of the cell.
Growth factor binds to what domain?
The ectodomain
Domain that passes the plasma membrane
Transmembrane domain
What domain is the region of homology located?
Cytoplasmic domain (inside the cell - shows high homology for Src)
Since the growth factor receptor shows high homology to Src, we now know that the growth factor receptor is also a(n)
tyrosine kinase
3 features of tyrosine kinase receptors (RTKs):
- All tyrosine kinase receptors have the same 3 functional domains.
- The kinase (cytoplasmic?) domain is highly conserved.
3.
Which proteins in the monkey kidney cells became phosphorylated?
Before growth factor addition, tyrosine phosphorylation levels were low. After, a lot of proteins became phosphorylated, especially proteins around plasma membrane.
Inhibitor of tyrosine kinase (inhibits phosphorylation)
AG1478
In absence of a growth factor, RTKs are
monomers (single peptides)
Since monomers can move freely on plasma membrane, they can bump into each other. In the presence of a growth factor, when RTKs bump into each other, the binding of the GF to the extracellular domain of the GF-R
Briefly increases affinity of the two receptors, causing dimerization; formation of a dimer (two peptides)
Two critical changes of RTKs following ligand binding (two important events of RTK activation):
- Dimerization
- Phosphorylation (transphosphorylation) of cytoplasmic domain
Two receptors aggregate together to form a complex
Dimer (dimerization)
Transphosphorylation
Kinase localized on one receptor, phosphorylate tails? on another receptor
Tumor virus that can cause tumor formation in birds
AEV (avian erythroblastosis virus)
AEV is similar to
RSV (rous-sarcoma virus)
Oncogene in AEV
v-EerbB
v-EerbB is similar to
Src
(think: AEV similar to RSV)
How can v-EerbB transform cells and cause cancer formation?
v-EerbB displays high homology to EGF-R; however, it lacks extra ectodomain (deletion)
The deletion of the ectodomain results in
conversion of the proto-oncogene into an oncogene
Need for ligand (i.e. growth factor) to bind to receptor for activation
Ligand-dependent firing
4 mechanisms to cause deregulation of receptor firing:
- deletion of ectodomain
- mutation (AA substitution)
- overexpression
- fusion of receptors
Dimerization and phosphorylation can become ligand-independent via receptor mutation, when
receptor is mutated (ex. deletion of extracellular receptor domain), the receptor behaves differently than normal
Dimerization and phosphorylation can become ligand-independent via mutation in any domain, by
substitution of amino acids
Dimerization and phosphorylation can become ligand-independent via overexpression, when
cell produces excessive amount of protein, so cell would contain numerous wild-type number of receptors, the cell would express excessive amount of the receptor, causing frequent collisions
Unrelated proteins covalently attack each other, forming
fusion protein
Fusion proteins can form
FIG proteins that can form a dimer (this is a problem because of abnormal activation)
Normal cells normally do not produce their own
ligands
3 major types of signaling:
Paracrine, autocrine, endocrine
signaling same cell; a form of signaling in which a cell manufactures its own mitogens
autocrine signaling
signaling long distance
endocrine signaling
signaling to neighboring cells
paracrine signaling
virus that affects cell division of mesenchymal cells (hint: it is a tumor virus)
Simian sarcoma virus
oncogene in Simian sarcoma virus
v-sis
T/F v-sis is similar to Src
true
cloning v-sis, showed its similarities to
PDGF
PDGF is a growth factor in
mesenchymal cells
A nuclear cell displays PDGF receptor, normal cell signaling division depends on (?) cells. When the cell is infected with Simian sarcoma virus, the cell will produce
v-sis protein ligand
v-sis protein ligand produced by infected cell can then bind to its own PDGF receptor, this is an example of
autocrine signaling (because cell produces its own “growth factor”, becoming autonomous)
autocrine signaling can lead to
deregulation of receptor firing
what number of proteins have general structures of the EGF-R and PDGF-R (in the human genome)
59
Deregulated activation caused by abnormal dimerization of RTKs (review):
Overexpression of receptors (dimerization via collision)
Mutation (AA substitution)
Deletion of ectodomain of RTKs
Fusion of RTKs to other proteins that dimerize
Ordered sequences of biochemical reactions inside the cell; with high specificity and speed
Signaling cascades
Light sensitivity/intensity unit of the eye (fruit fly); each is formed by a series of seven cells
Ommatidia
Gene that encodes a homolog of the FGF-R (a RTK) in ommatidium of fruit fly - so that it only contains 6 cells
Sevenless (homolog of RTK)
functions downstream of the sevenless and activates Ras
Sos (son of sevenless)
Sos in fly =
GEF (guanine nucleotide exchange factors; in yeast)
GEF affects
activity of guanine nucleotide binding protein (Ras)
Ras is a
signaling molecule that can switch between active/inactive; its activation state is determined by binding of guanine nucleotide
inactive form of Ras
binding GDP
active form of Ras
binding GTP
Sos can activate GEF, which converts the guanine nucleotide from
GDP to GTP (inactive to active Ras)
Molecule found in gap between sevenless and sos
Grb2
Signaling cascade upstream of Ras
RTK -> Grb2 -> Sos -> Ras
Signaling cascade upstream of Ras (for adaptive proteins)
RTK -> Shc -> Grb2 -> Sos -> Ras
Another molecule that can be found in gap between sevenless and sos (depending on type); adaptive proteins
Shc
RTKs affect physical location of downstream components without changing their intrinsic activity
Localization model
Two theoretical ways receptor can pass signal (activate) to downstream molecule
- activation (of surrounding molecules) - changing biochemical property of molecules, in presence of GF; phosphorylation – this was proved incorrect
- increase affinity of receptor by phosphorylation to attract molecules (in presence of GF); not changing biochemical property, but physical location – this is correct (localization model)
Src protein contains 3 domains:
SH1 (catalytic domain, (?) ATP)
SH2 (binding to pY-containing peptide)
SH3 (binding to proline-rich sequence domain)
A typical SH2 domain structure contains
100 amino acid residues
Structure of SH2
assembled from a pair of antiparallel beta-pleated sheets surrounded by alpha-helices
Sites on SH2 are responsible for what?
bind to phosphorylated tyrosine (pY); highly related to signaling transduction; can also bind to 3-6 amino acid (peptide) that follows pY
Function of SH2 domain
functions as modular plug, recognizing both pY and side-chains of AA (3-6) that flank the pY on its C-terminal side
Human genome is estimated to encode at least ___ distinct SH2 groups.
120
pY can attack proteins/substrates that contain
(proteins that are substrates of PDGF-beta-R (ex. src, GAP, SHP2, etc))
SH2
the 3-6 amino acid residue determines what for pY
where it can bind on the proteins/substrates
Why does one type of receptor (PDGF-R) recruit certain types of proteins and other receptors (EGF-R) recruits other types of proteins?
Each receptor contains unique residues (specificity)
Grb2 and Shc are what kind of proteins
Bridging proteins
Ras intrinsic activity; hydrolyzes GTP to GDP
GTPase
GTPase-activating proteins
GAP
3 effector molecules downstream of Ras
PI3K, Raf, Ral-GEF (each have different signaling pathways)
Activated Ras attracts Raf from cytosol, the binding of Raf to Ras leads conformational changes of Raf;
Deregulation of this pathway contributes to certain cancer phenotypes (loss of contact inhibition, anchorage dependence, etc);
Deregulation can contribute to some cancers.
MAPK (mitogen-activated protein kinase) pathway
Steps in MAPK pathway
Ras -> Raf -> MEK -> Erk 1 or 2 -> Mnk1 (and other proteins) -> elF4E (protein synthesis)
cyclin D1; protein that drives cell proliferation
Ets
Ras -> PI3K -> PIP3 -> Akt/PKB (or Rho-GEFs) -> Bad inhibition of apoptosis / mTOR stimulation of protein synthesis (cell growth) / GSK-3beta stimulation of cell proliferation
PI3 kinase pathway steps
Functions to synthesize phosphatidylinositol (3,4,5) triphosphate (PIP3)
PI3K (phosphatidylinositol 3-kinase)
A small minority of head groups in phospholipid bilayer contain ____ sugars
inositol
Phospholipid structure + attach inositol sugar =
phosphatidylinositol (PI)
3 components of PI (phosphatidylinositol):
- 2 fatty acids with long hydrocarbon tails
- glycerol
- phosphate and inositol
PI kinases catalyzes the transition between
PI and PIP2
(phosphatidylinositol to phosphatidylinositol-4,5-diphosphate)
PI3 kinases (PI3K) then leads PIP2 to become
PIP3 (phosphatidylinositol-3,4,5-triphosphate)
PIP3 (binding site) attracts and activates which two molecules
Akt/PKB and Rho-GEF
A pleckstrin homology (PH) domain-containing protein
Akt/PKB
Functions of Akt/PKB:
- inhibit apoptosis
- stimulate cell division
- promote cell growth
PIP3 formation/synthesis is controlled by which two proteins
PI3K and PTEN
tumor suppressor protein in PIP3 formation/synthesis
PTEN
PIP3 serves as docking site for
Akt/PKB (once they bind, Akt/PKB becomes phosphorylated and activated)
T/F: Phospho-Akt promotes cell growth
True
In mouse pten-/- epithelial cells (immunostained with antibody PTEN antibody), what happened to the cells?
Tumor cells grow because PTEN is lost
In mouse pten cells immunostained with an anti-phospho-Akt/PKB-antibody, what happened to the cells?
There is abundance of PPI3
Signaling pathways have what sort of relationship?
Interconnected
T/F: Signaling pathways are linear
False
Anti-growth genes
Tumor suppressor genes
T/F: Oncogenes are more important than tumor suppressor genes.
False, just as important would be correct
Prevalence of retinoblastoma
1 in 20,000 children (rare)
Retinoblastoma age of diagnosis
birth to 6-8 years
Two types of retinoblastoma:
unilateral and bilateral
Affect only single eye
Unilateral retinoblastoma
Affect both eyes
Bilateral retinoblastoma
What form are unilateral retinoblastomas?
Sporadic form
What form are bilateral retinoblastomas?
Familial form
Rate of incidence with respect to time after retinoblastomas diagnosis:
36% bilateral at 50 years
5.69% unilateral at 50 years
One hit and two hit theories were studied when
1971
Bilateral retinoblastomas requires __#__ change(s)
1
Each hit was presumed to represent what kind of mutation?
somatic
In sporadic retinoblastoma, one somatic mutation leads to
one mutant Rb allele
In familial retinoblastoma, one somatic mutation (one hit) leads to
two mutant Rb gene copies (tumor formation)
In sporadic retinoblastoma, how many mutation events must occur to have two mutant Rb gene copies?
2 somatic mutations (two hit for tumor formation)
Both Rb genes inactivated =
no functional genes (tumor formation)
Occurence of a single mutation on a single gene
1 x 10^-6 per generation
Occurence of a double mutation on a single gene
1 x 10^-12 per generation
The first mutation is what kind of event
sporadic mutation
The second mutation is what kind of event
loss of heterozygosity (LOH)
Recombination occur during cell proliferation
Mitotic recombination
Also called “allelic deletion”; a genetic alteration that converts a chromosome region from heterozygous to homozygous
Loss of heterozygosity (LOH)
Which occurs more sporadic mutation or mitotic recombination?
Mitotic recombination occurs 10-100x more often than sporadic mutation
Which occurs more loss of heterozygosity or mitotic recombination?
Loss of heterozygosity occurs more frequently than mitotic recombination
Frequency of events (in order)
LOH > methylation > somatic mutation
- DNA poly begins replication on template strand of red chromosome
- DNA poly jumps to template strand of homologous, green chromosome
- after copying segment of green template strand, DNA poly jumps back to template strand of red chromosome and continues copying
Gene conversion and LOH
- Initial heterozygosity at Rb locus
- Nondisjunction at mitosis
- Subsequent loss of extra chromosome
Chromosomal nondisjunction and LOH
What chromosome region is deleted in retinoblastoma patients?
Chromosome 13, 2nd and 4th bands of the 1st region on the long (q) arm of chromosome 13; 13q12-13q14
What year were mutations of the Rb gene in retinoblastomas discovered?
1986
The molecular governor of the R point transition in cell cycling
Rb
Unphosphorylated Rb binds to the E2F, and this binding prevents E2F from…
activating the transcription of genes coding for proteins required for DNA replication in S phase
In cells stimulated by growth factors, what phosphorylates the Rb protein?
activated CDK-cyclin
Can phosphorylated or unphosphorylated Rb bind to E2F?
Phosphorylated Rb cannot. Unphosphorylated Rb can.
Number of tumor suppressor genes:
> 50
Important mechanism in shutting down tumor suppressor genes; covalently attach methyl group to a cytosine base; heritable, reversible
DNA methylation
DNA methylation steps
- covalently attach methyl group to cytosine base
- in mammalian cells, methylation is found only when cytosines are located in a position that is 5’ to guanosines: MeCpG
- When MeCpG occurs in the vicinity of a gene promoter, expression of gene can be repressed
Important mechanism in inactivating tumor suppressor genes in tumors
Promoter methylation
The study of heritable changes in gene function that occur without a change in the DNA sequence
Epigenetics
One copy is methylated, the second is lost through
LOH
DNA methylation and LOH work together to
shut down suppressor genes
Colonic polyps are common in what group of people
Very common in age 70+
What percentage of colon cancer is sporadic?
> 95%
Carpet of hundreds of small polyps in the colon; finger-like projections
Familial adenomatous polyposis (FAP)
Contributes to cell proliferation; controls cancer formation (colon cancer and others) by controlling abundance of B-catenin protein
Wnt-B-catenin pathway
tag phosphorylated B-catenin for degradation
Apc
Why cells can’t divide in absence of Wnt
low B-catenin
controls cells division; acts like growth factor
(in Wnt-B-catenin pathway)
Wnt
In a normal cell, B-catenin is
synthesized then phosphorylated then degraded
Phosphorylates B-catenin, so that Apc can identify and tag it for degradation
GSK-3B (glycogen synthase kinase-3B)
When GSK-3B is inactive/enzymatically dead,
Apc can not tag B-catenin for degradation, so B-catenin accumulates and is in abundance
Binding of Wnt to the frizzled receptors causes inhibition of
GSK-3B, preventing phosphorylation and degradation of B-catenin
B-catenin associates with ____ in the nucleus and drives cell proliferation
TF Tcf/Lef
B-catenin is similar to myc, playing a role in cell proliferation, except it is not a:
transcription factor
Wnt is secreted by
stroma cells
In mutated/deregulated colonic crypts, Apc protein is defective, B-catenin levels remain high in absence of intense Wnt signaling. Cells stop migrating upward, accumulate within crypts, and ultimately generate a
adenomatous polyp (colon polyp)
Stem cells receive Wnt signal from stroma. B-catenin interacts with Tcf/Lef and promotes proliferation in stem cells. When cells move upward, stimulation by Wnts decreases, leading to increased degradation of B-catenin. Cells enter apoptosis after 3-4 days
normal cells (colon polyp)
Apc structure
contains multiple protein binding domains, and the gene encoding Apc is frequently mutated
mutations that affect B-catenin binding
germ-line mutations and somatic mutations (proving function of Apc)
3 major suppressor types:
- PTN: convert PiP3
- PRB: control cell proliferation; control transcription
- Acp: target B-catenin for degradation
2 types of anti-growth genes
gatekeepers and caretakers
directly control biology of cells by affecting how they proliferate, differentiate, or die
gatekeepers
control biology of cells through maintenance of cellular genomes
caretakers
gatekeepers are
tumor suppressor genes
a protein neckwork (signal processing circuit) that receives signals from various sources outside and inside the cells, integrates them, and decides the cell’s fate
cell cycle clock
Where is the cell cycle clock located?
nucleus
4 phases of mammalian cell cycle
M, G1, S, G2
nuclear division and cytoplasm division; ~ 1 hr
mitotic phase
G1 phase lasts approximately
12-15 hrs
S (synthesis) phase lasts approximately
6-8 hrs
G2 phase lasts approximately
3-5 hrs
can’t see DNA under normal light microscope (need electron microscope)
interphase
DNA visible under light microscope.
Mitotic spindle will assemble; centrosome moves in opposite direction
prophase
Chromosome displayed central in dividing cell
metaphase
Chromatids move in opposite direction
anaphase
Chromatids reached the 2 poles.
Nuclear membrane becomes reformed (was fragmented)
telophase
Interphase consists of
G1, S, G2
Karyotype of normal cells, replicate how many times
once
Karyotype of Rad17-deficient cells
malfunction/no function -> continue replicating
point where cell makes decision to advance through remainder of cell cycle through M phase, to remain in G1, or retreat from active cell cycle to G0
restriction point
before and at the restriction point, cell pays attention to
environment
regulatory subunits of heterodimeric protein kinases that control cell cycle events
cyclin
a group of serine/threonine kinases that are involved in regulation of cell cycle; push from one phase to another via phosphorylation; dependent on cyclin
CDKs
cyclin-CDK complexes depend on
the association of a specific cyclin type having a physical interaction with CDK
cyclin A + CDK increases catalytic activity of CDK2 by ____ fold
400,000
types of cyclin
A, B, D, E
3 types of CDK
CDC2, CDK4/6, CDK2
cyclin B pairs with
CDC2 in M phase
cyclin D pairs with
CDK4/6 in G1 phase to R point
cyclin E pairs with
CDK2 from R point to S phase
cyclin A pairs with
CDK2 for first 1/2 of S phase
CDC2 for second 1/2 of S phase and whole G2 phase
cell cycle progression depends on changes in levels and availability of
cyclin during the various phases
Fluctuation of cyclin B, E, and A levels during cell cycle in most mammalian cells
are tightly coordinated with schedule of advances through various phases
Extracellular signals (especially mitogenic growth factors) strongly influence levels of cycle type
D (can sense/respond to environmental conditions)
D-type cyclins serve to convey signals from extracellular environment to the
cell cycle clock in the cell nucleus
there are various pathways involved in control of cyclin type ____ expression
D1
What percentage of the cell cycle is influenced by extracellular signals and automatically programmed?
80-90% of G1 = extracellular signals
10-20% of G1, S, G2, M = automatic
Cyclin-CDK complexes are regulated by ____; a group of proteins that affect activities of cyclin-CDK complexes
CDK inhibitors (CDKIs)
Seven important CDKIs (tumor suppressor proteins)
4 INK4 (inhibitors of CDK4); affect only CDK4/6
3 other affect CDK2 and CDC2 (CDK4/6)
TGF-B can inhibit cell division via
expression of p15^INK4B and weakly, p21^Cip1
DNA damage causes rapid increases in
p21^Cip1
blot technique to detect mRNA
northern blot
molecular governor of R point transition
pRb
highly phosphorylated Rb; happens around restriction point
hyperphosphorylation
weakly phosphorylated
hypophosphorylation
CDK4/6 converts
unphosphorylated to hypophosphorylated
phase at which E2F is released
late G1, after hyperphosphorylation at R point, pRb releases E2Fs, allowing E2Fs to function as TFs
pRb binds to E2Fs, blocking transcription activated domain
early/mid G1 when unphosphorylated/hypophosphorylated
As cells enter into S phase the E2Fs are
inactivated and/or degraded
