p53 and ApoptosisChapter 9
p53 discovered from association with SV40 Large T-antigen.
Mouse fibroblast 3T3 cells transformed with SV40 virus
F9 mouse embryonal carcinoma cells
-grown in presence of S35 methionine
Proteins Immunoprecipitated by normal or SV40 reactive
antiserum detected by autoradiography.
What is the arrow pointing to?
TP53 is the gene name for gene coding for p53 protein
Mutations in TP53 seen in more than 50% of all
human cancers.
Is p53 an oncogene or a tumor suppressor?
Does introduction of p53 along with ras
oncogene increase foci formation?
Loss of p53 in mouse germ line reduces
survival of mice due to development of
sarcomas and leukemia
Most common mutations in p53 were
missense mutations that affected its
ability to bind to DNA
How does p53 function?
It’s a DNA binding protein that can tetramerize (4) to itself
Dominant Negative mutations in p53
Guardian of the Genome!
Integrates information from
toxic signals to result in cell
cycle arrest/DNA repair or
apotosis
What do these results tell you about how p53 brings about cell cycle arrest in
response to radiation?
Control of p53 protein levels
Mdm2 acts as a p53 inhibitor
keeping levels of p53 low by
targeting it for degradation by
proteasome machinery
Response to DNA damage results in
stabilization of p53 levels
p53 phosphorylation by Chk1/2 inactivates
Mdm2 and removes it from p53 thereby
activating it.
Note: Negative feedback loop!
Inhibitor of an inhibitor is an activator!
ARF is a tumor suppressor like p53
Targets of p53
What makes these factors pro or anti-apotosis?
How does p53 activate apoptosis?
Can you spot at least 3
different effects of p53
on apoptotic pathway?
Advantages to cancer cells due to loss of p53 function
Inactivation of p53 affects multiples steps in tumor progression:
Oncogenes like myc trigger p53 dependent apoptosis. So loss of p53=growth advantage for transformed cells
Anoxia: loss of p53 allows for oxygen independent growth
DNA damage agents-increased mutability, activates oncogenes, suppresses tsgs
50% of tumors carry mutations in p53.
Many of the remaining tumors cause ARF loss or Mdm2 overexpression.
Anti-apoptotic oncogenes are in red and show
increased expression in cancer cells
Proapoptotic TSGs are in blue and show
decreased expression in cancer cells.
You don’t need to know details of all the steps.
Cell Immortalization and
Tumorigenesis
The Immortal Life of Henrietta Lacks
• Replicative life span: The
number of cell divisions a cell
can undergo before
dying/senescence
• Senescence: Cells that are
metabolically active but have
irreversibly lost their ability to
enter the cell cycle. Not the
same as quiescent state (G0)
• Immortalization: process by
which a normal cells acquires
the ability to replicate
indefinitely
Senescent cells
• Cells stop growing after a
predetermined number of cell
divisions.
• Acquire a large cytoplasm =fried egg
appearance.
• Require appropriate growth factor to
maintain viability in cell culture.
• They express growth factor receptors
on their surface but do not
proliferate.
• Why?
What causes senescence?
• Replicative life span of cultured cells
depends on originating species, cell type
and age of donor organism.
• Regulator of lifespan
synthesized early in development
and not after (ES stem cells have unlimited
life span)
Is present at high concentrations in
embryonic cells
is progressively diluted by each cell
division
• Other explanations: ROS damage
Physiological Basis for Replicative Senescence
Inhibitors of cell cycle
progression
Normal
Senescent cells
Ectopic
expression
of p16INK4A
Orange=actin stress
fibers, yellow=focal
contact with substrates
Lower oxygenation levels=increase in replicative lifespan
High oxygen=high oxidative damage due to
ROS=oxidative induced lesions in chromosomal
DNA
Cell culture media that prevent induction of p53
and p16INK4A postpone replicative senescence
SV40 T antigen and Replicative Senescence
• Recall the large T antigen
has the ability to bind and
sequester both Rb and p53.
• Does loss of these tumor
suppressor genes allow
cancer cells to overcome
senescence?
• p53 and Rb mutations
account for a majority of
human cancers.
Does Senescence occur in vivo?
Formation of
Senescence
associated
heterochromatin foci
(SAHF)=gene
expression silenced.
Staining for senescent cell
expression of β-galactosidase
Yes-but is harder to detect!
Immortalization requires loss of senescence
and overcoming crisis
• Measures of Physiological stress= induces senescence if damage is
too high. Cells in Senescence=reasonably stable karyotype
• Cell doubling clock=measures how many doublings have occurred and
leads to crisis if doubling number exceeds a set limit for the cell
lineage. Cells in crisis=widespread karyotypic disarray. Crisis leads to
apoptotic cell death.
• For cells to be immortal, they need to overcome both regulators of
physiological stress and cell cycle doubling
Cell Doubling Clock=
Telomeres!
Normal cells at metaphase
TRF-/- cells at metaphase
• In Chromosomal DNA not in
proteins/metabolites.
• Stable passed on from one
generation to another
• But how does it count?
Yellow/green dots=telomeres
red= chromosomes
The telomerase holoenzyme is a RT carrying
its own template RNA
molecule.=ribonucleoprotein
In normal somatic cells, telomerase levels
are low due to low expression of hTERT
protein
Telomerase is able to use the internal RNA
molecule to direct extension in the 5’ to 3’
directions
Telomeric repeats of many kilobases seen
at the end of chromosomes
Telomere length and Crisis
Southern Blotting with Genomic DNA treated with
restriction enzymes that do not cut in the telomeres
and probed with a probe specific for the telomeres
Telomeres get progressively shorter till they reach
critical length that triggers crisis
Mechanisms of Breakage-fusion-bridge cycles
Chromosomes in crisis-lose telomeres on one
end more often than the other.
The double stranded breaks in DNA ends are
repaired by end to end fusion generating a
dicentric chromatid with two telomeres and
one shared centromere.
In Anaphase, these dicentric chromosomes
form bridges in the middle of mitotic spindle
The dicentric chromatid ripped apart at weak
points generating a telomere less end that can
now go through another round of this cycle
MAJOR CHANGES IN KARYOTYPE=often seen in
cancer cells that enter and escape from crisis!
Cancer cells upregulate levels of telomerase
Measures the activity of cellular
telomerase by providing an oligo
template
Levels of hTERT mRNA induced-more telomerase is made in immortal cells
hTR RNA levels do not change
How does overexpression
of hTERT affect telomere
length and replicative life
span?
Expression of dominant-negative
hTERT sequesters other
components of the telomerase
holoenzyme and prevents its
function.
What is the phenotype of its
overexpression in cancer cell
lines?
Inhibitors of telomerase
activity-effective anticancer drugs?
Tumorigenesis is a multi decade process
Cancer incidence increases with age-multiple events need to take place before a tumor can be detected.
Tumor Progression can be followed by histopathology
It is not a linear pathway in all cancer types.
Some of the stages may be skipped
Are some steps in this progression dead ends?
Clinical evidence for tumor progression
Carcinoma arising out of adenoma!
Molecular events that underlie tumor progression
DNA from various samples at different stages of colon cancer analyzed for loss of heterozygosity
Can be correlated to loss of tumor suppressor genes(TSG)
The multiple molecular mistakes that need to occur before tumor progresses into cancer!
Metastasis and Invasion
Metastases
Primary tumors: Formed at the site of the initial uncontrolled proliferation event
• Cancer cells are contained within tissue boundaries
• Can grow to significant sizes in certain organs without affecting function
• Uncontrolled growth of primary tumors compromise functioning of organpassage of metabolites, fluids, airways may be affected by primary tumor
growth at primary site
• Only responsible of 10% cancer deaths
Metastases: Cancerous growths at tissue locations far away from the site of
primary tumor
• Retains tissue characteristic of originating tissue
• Utilize the circulatory systems of the body-blood/lymph to disseminate
• Exhibit tissue preference for metastatic growth: tropism
• Primary tumors arising in certain cells/tissues have a higher tendency to
metastatize: melanomas have very high metastatic rate
• Micrometastases are already formed by the time primary tumor is diagnosed
Non-Hodgkin’s lymphoma
patient showing multiple sites
of aerobic glycolysis in the
abdominal region on PET/CAT
scan using radiolabeled
fluorodeoxyglucose
Invasion-Metastasis Cascade
Commonly seen in epithelial cells giving rise to carcinomas.
Most of this lecture is based on these cell types.
But, cancers originating in connective and nervous tissues follow a
similar pathway.
Important to know the stages: what is happening during each of the stages
Seven steps to metastases development
• Primary tumor: Localized growth within
tissue boundaries
• Localized invasion: Crossing basement
membrane
• Intravasation: Crossing the endothelial cell
lining into blood vessels
• Circulatory tumor cells: Cancer cells in
circulation in blood/lymph
• Trapping in the blood vessels of secondary
sites
• Extravasation: Crossing the endothelial
lining out of blood vessels into tissue
• Colonization: Micrometastases grow into
macrometastases
Epithelial tissue arrangement
Epithelial cells separated from underlying stromal cells (connective tissue) by a thin
Extracellular matrix (ECM) called basement membrane. For cancerous epithelial cells to
develop into carcinomas, they have to invade the basement membrane and pass through
the stromal layer.
Metastasis and Invasion
Metastases
Primary tumors: Formed at the site of the initial uncontrolled proliferation event
• Cancer cells are contained within tissue boundaries
• Can grow to significant sizes in certain organs without affecting function
• Uncontrolled growth of primary tumors compromise functioning of organpassage of metabolites, fluids, airways may be affected by primary tumor
growth at primary site
• Only responsible of 10% cancer deaths
Metastases: Cancerous growths at tissue locations far away from the site of
primary tumor
• Retains tissue characteristic of originating tissue
• Utilize the circulatory systems of the body-blood/lymph to disseminate
• Exhibit tissue preference for metastatic growth: tropism
• Primary tumors arising in certain cells/tissues have a higher tendency to
metastatize: melanomas have very high metastatic rate
• Micrometastases are already formed by the time primary tumor is diagnosed
Non-Hodgkin’s lymphoma
patient showing multiple sites
of aerobic glycolysis in the
abdominal region on PET/CAT
scan using radiolabeled
fluorodeoxyglucose
Invasion-Metastasis Cascade
Commonly seen in epithelial cells giving rise to carcinomas.
Most of this lecture is based on these cell types.
But, cancers originating in connective and nervous tissues follow a
similar pathway.
Important to know the stages: what is happening during each of the stages
Seven steps to metastases development
• Primary tumor: Localized growth within
tissue boundaries
• Localized invasion: Crossing basement
membrane
• Intravasation: Crossing the endothelial cell
lining into blood vessels
• Circulatory tumor cells: Cancer cells in
circulation in blood/lymph
• Trapping in the blood vessels of secondary
sites
• Extravasation: Crossing the endothelial
lining out of blood vessels into tissue
• Colonization: Micrometastases grow into
macrometastases
Epithelial tissue arrangement
Epithelial cells separated from underlying stromal cells (connective tissue) by a thin
Extracellular matrix (ECM) called basement membrane. For cancerous epithelial cells to
develop into carcinomas, they have to invade the basement membrane and pass through
the stromal layer.
Epithelial Mesenchymal Transition (EMT)
It is not birth, marriage or death, but gastrulation, which is the
truly the most important time of your life-Lewis Wolpert,
embryologist, 1986
EMT associated with multiple stages of embryonic
development: formation of mesoderm, neural crest cells etc.
Also seen during wound healing process.
Cancer cells reactivate this latent behavioral program for their
own gain.
• To invade, epithelial cells must:
• Shed epithelial phenotypes (morphology, gene expression
patterns)
• Detach from sheets
• Assume the shape and gene expression characteristics of
mesenchymal cells
• EMT allows formerly epithelial cells to attain migratory
capabilities
Epithelial cells of sea urchin embryo
delaminate and move towards the
interior of embryo
Maintenance of epithelial structure requires E-cadherin
E-cadherin belongs to the Integrin class of receptors that interact with ECM proteins and components
E-cadherin molecules on neighboring cells are linked together on their extracellular side to form
adherens junctions.
On the cytoplasmic side, E-cadherin is linked to actin cytoskeleton through β-catenin (anchor protein)
Loss of E-cadherin linked to loss of epithelial state
• In invasive human carcinomas, CDH1 gene
(coding for E-cadherin protein) is subject to
increased promoter methylation, leading
to repression of transcription of CDH1.
• Overexpression of E-cadherin in carcinoma
cells suppressed invasiveness and
metastatic dissemination.
• Loss of E-cadherin is sufficient to trigger
EMT.
• Loss of E-cadherin releases free β-catenin
that can translocate into nucleus and
activate transcription of genes involved in
EMT program.
Metastasis and Invasion
Metastases
Primary tumors: Formed at the site of the initial uncontrolled proliferation event
• Cancer cells are contained within tissue boundaries
• Can grow to significant sizes in certain organs without affecting function
• Uncontrolled growth of primary tumors compromise functioning of organpassage of metabolites, fluids, airways may be affected by primary tumor
growth at primary site
• Only responsible of 10% cancer deaths
Metastases: Cancerous growths at tissue locations far away from the site of
primary tumor
• Retains tissue characteristic of originating tissue
• Utilize the circulatory systems of the body-blood/lymph to disseminate
• Exhibit tissue preference for metastatic growth: tropism
• Primary tumors arising in certain cells/tissues have a higher tendency to
metastatize: melanomas have very high metastatic rate
• Micrometastases are already formed by the time primary tumor is diagnosed
Non-Hodgkin’s lymphoma
patient showing multiple sites
of aerobic glycolysis in the
abdominal region on PET/CAT
scan using radiolabeled
fluorodeoxyglucose
Invasion-Metastasis Cascade
Commonly seen in epithelial cells giving rise to carcinomas.
Most of this lecture is based on these cell types.
But, cancers originating in connective and nervous tissues follow a
similar pathway.
Important to know the stages: what is happening during each of the stages
Seven steps to metastases development
• Primary tumor: Localized growth within
tissue boundaries
• Localized invasion: Crossing basement
membrane
• Intravasation: Crossing the endothelial cell
lining into blood vessels
• Circulatory tumor cells: Cancer cells in
circulation in blood/lymph
• Trapping in the blood vessels of secondary
sites
• Extravasation: Crossing the endothelial
lining out of blood vessels into tissue
• Colonization: Micrometastases grow into
macrometastases
Epithelial tissue arrangement
Epithelial cells separated from underlying stromal cells (connective tissue) by a thin
Extracellular matrix (ECM) called basement membrane. For cancerous epithelial cells to
develop into carcinomas, they have to invade the basement membrane and pass through
the stromal layer.
Epithelial Mesenchymal Transition (EMT)
It is not birth, marriage or death, but gastrulation, which is the
truly the most important time of your life-Lewis Wolpert,
embryologist, 1986
EMT associated with multiple stages of embryonic
development: formation of mesoderm, neural crest cells etc.
Also seen during wound healing process.
Cancer cells reactivate this latent behavioral program for their
own gain.
• To invade, epithelial cells must:
• Shed epithelial phenotypes (morphology, gene expression
patterns)
• Detach from sheets
• Assume the shape and gene expression characteristics of
mesenchymal cells
• EMT allows formerly epithelial cells to attain migratory
capabilities
Epithelial cells of sea urchin embryo
delaminate and move towards the
interior of embryo
Maintenance of epithelial structure requires E-cadherin
E-cadherin belongs to the Integrin class of receptors that interact with ECM proteins and components
E-cadherin molecules on neighboring cells are linked together on their extracellular side to form
adherens junctions.
On the cytoplasmic side, E-cadherin is linked to actin cytoskeleton through β-catenin (anchor protein)
Loss of E-cadherin linked to loss of epithelial state
• In invasive human carcinomas, CDH1 gene
(coding for E-cadherin protein) is subject to
increased promoter methylation, leading
to repression of transcription of CDH1.
• Overexpression of E-cadherin in carcinoma
cells suppressed invasiveness and
metastatic dissemination.
• Loss of E-cadherin is sufficient to trigger
EMT.
• Loss of E-cadherin releases free β-catenin
that can translocate into nucleus and
activate transcription of genes involved in
EMT program.
EMT occurs primarily at the invasive front of cancer
• EMT occurs primarily at the
interface of tumor epithelium and
stroma.
• Only cells on the outer edge of the
tumor undergo EMT.
• Cells away from the edge retain Ecadherin or cytokeratin =epithelial
state
• Cells closer to the edge=loss of Ecadherin or acquisition of vimentin
=mesenchymal state
Green=E-cadherin
Red= β-catenin
Overlap=yellow
Experimentally
transformed MECs
implanted in
immunocompromised
mouse.
Human cytokeratin=red
Human vimentin=green
Stromal cells induce EMT in epithelial cells
• Reactive stroma=stromal cells having to
adapt to the presence and growth of
primary tumor. Accumulates
inflammatory cells like macrophages
• Mediated through release of TGF-β by
stromal cells.
• TGF-β loses its cytostatic effect in cancer
cells due to loss of pRb signaling
• Removal of TGF-β from cancer cells that
have undergone EMT reverses EMT
EpRas tumor cells carrying ras oncogene
Recruitment of macrophages into reactive stroma key to invasiveness
Secretion of CSF-1 by carcinoma cells in cancer prone
transgenic mice results in recruitment of large numbers of
tumor associated macrophages (TAMs) into stroma.
These TAMs secrete large quantities of growth promoting
factors such as EGF.
Macrophage-tumor cell interaction key to invasiveness. In the Op-/- mutants do not secrete
Colony Stimulating Factor:
absence of CSF-1 secretion, tumors do not break through
CSF-1
basement membrane
In the presence of macrophages,
carcinoma cells acquire the ability to
invade into collagen gel layer
Stromal cells lead the way
• Reactive stromal cells secrete many
proteases that cleave proteins of ECM
and clear a path for the invasive cancer
cells to make their way through the
stroma
Tumor cells in blue
Macrophages in green
Secrete cathepsin (protease) in
yellow to clear the way
Intravasation
• Much less is known
• Requires action of cancer cells,
macrophages and endothelial cells
(tumor microenvironment of
metastasis)
• Somehow this triad of cells allows
carcinoma cells to breach endothelial
lining of capillaries
• Increased number of triads= poor
prognosis
Staining for presence of all three cell types
Spread through circulatory system
• Very few cancer cells can survive in
circulation 1in 10000.
• Many lost due to hydrodynamic shear
forces
• Survival of cancer cells enhanced by
association with platelets
• CTCs counts=cancer markers?
Cancer cells=green
Hematopoietic cells=red
• Cancer cells trapped in lung capillary
beds=primary site of metastases for many
cancers
Extravasation
• Cells squeeze through endothelial walls
• Or grow inside the capillary eventually
blocking it, bursting through the
endothelial cell lining and basement
membrane
Cancer cells undergo a mesenchymal-epithelial
transition (MET) on extravasation
Tropism
• Which tissue is the site for metastatic
growth
• Depends on cell surface receptors on
cancer cells and the secondary tissue
they enter
• Therapeutic potential?
Seed and soil hypothesis
Depends on ability of tumor to colonize
and grow in the new tissue.
Depends on circulatory route
Ability to colonize=acquired
specialization=additional mutations.
Ease of acquiring these mutations decides
tropism
Width of arrow indicative of tissue preference
for metastases
Micrometastases to Macrometastases
• Colonization=ability to grow in the new
tissue site to form macrometastases
• Most micrometases die rapidly or do not
grow due to lack of familiar growth factors
and tissue environment
• More than 30% of women diagnosed with
primary breast cancer already harbor
thousands of micrometastases in bone
marrow. Only 50% of these patients go on to
develop metastatic cancer of the bone
• Low conversion rate=metastatic insufficiency
• Responsible for variation in metastatic rates
among different cancers
Anti-cytokeratin antibody
staining to detect presence
of epithelial cells in
mesenchymal populations
Know the basis of immune system
• Humoral response: antibody
production
• Cellular response: difference
between innate, adaptive
• Function of macrophages, dendritic
cells, NK cells,
• Role of antigen presentation in
priming adaptive responses, T-helper
cells
Immunosurveillance
These are not cancers of viral origin-cancer
cells likely to have only self antigens. So
how does the immune system recognize
and destroy cancer cells?
Ovarian carcinoma
patient survival rates
strongly correlated
with number of TILs.
Tumor infiltrating lymphocytes(TILs) recruited to cancer-immunosurveillance
Cancer characteristics that trigger immune
response
• Expression of tumor transplantation
antigens (TATAs) attract attention from
immune system. Expression of these
antigens is not limited to malignant cells.
• Increased expression of certain antigens-like
EGF
• Differentiation antigens: Lineage specific
proteins that are usually restricted in normal
cells.
Melanoma patients who trigger a strong
immune response to the presence of TATAs
have increased survival.
Immunoevasion by cancer cells
• If cancer cells can avoid detection by
immune system, then they cannot be
killed
• Reducing expression of TATAs-they are not
associated with survival fitness of cancer cells.
• Repress expression of certain classes of MHC
Class I proteins. Selective suppression needed
to evade attack by NK cells that look for cells
that do not have any MHC on their surface
• Reducing expression of stress signaling
proteins on cell surface-that are normally
recognized by NK cells to trigger cytotoxic
activity
Cancer cells attract T-regulatory cells
• T-regs kill cytotoxic Tcells that recognize the
same antigen
• In normal cells, Tregs
only 5-10% of CD4+
lymphocytes
• In cancer patients,
increases to 25-30%
• Increased presence of
T-regs is due to release
of T-reg attracting
chemokines (CCL22)
by tumor cells.
Sabri Abu Salim
100696781
BMS 673
Problem Set 1
1. How is the tsRSV different from the wild-type RSV virus? What is the cause of the
difference?
Temperature-sensitive mutants of RSV cause the cell to exhibit a transformed morphology
under a permissive temperature, unlike wild-type RSV viruses. This is because wild-type
RSV viruses are naturally occurring, non-mutated strains of the virus.
2. Under what conditions is the tsRSV able to transform the chicken embryo fibroblast.
When a temperature-sensitive mutant of RSV is injected into a fibroblast of a chicken embryo
then cultured at a permissive temperature of 37°C, the morphology of the cell was
transformed. However, when the temperature of the culture was raised to a non-permissive
temperature of 41°C, the morphologically transformed cell resulted in a non-transformed
morphology. Later, when the temperature was dropped to the permissive temperature, the
cells’ morphology was altered once more.
3. How do these results allow you to conclude that “RSV is needed for generating and
maintaining a transformed phenotype”?
The absence of the altered morphology due to a temperature increase revealed that this
phenotype requires the continued action of some temperature-sensitive viral protein. The
regaining of the altered morphology after many days of temperature reduction demonstrated
that the viral genome was still present in these cells at high temperatures despite their usual
appearance.
4. Why wouldn’t you be able to make the same conclusions from a similar experiment
with wild-type RSV?
The cells transformation will be genotypic; hence no observation will be possible, like in the
case of a temperature-sensitive mutant of RSV that causes a phenotypic cell transformation.
5. How is the mutagenic and carcinogenic ability of Compound X measured?
The Ames test is used to determine mutagenicity.
The Ames test allows us to quantify the mutagenic potential of our test chemical x. first, the
rat’s liver or another animal is homogenized to release enzymes. After that, the liver
homogenate is mixed with the test compound x, which frequently results in the test
compound x being metabolically converted to a chemically active, mutagenic state by the
liver enzyme. The solution, which still contains the liver homogenate, is placed in a dish of
mutant salmonella bacteria, which requires the Proliferation of the amino acid histidine in
their culture medium. Since histidine is absent in the medium, just microbes that have been
transformed to a histidine-independent genotype and phenotype will actually manage to
blossom, and every one of these will result in a vast colony that can be counted with the
unaided eye, showing the short openness created the number of changed microorganisms to
the stimulated compound x.
Other mutagenicity assays aid in determining potential carcinogenicity.
Sabri Abu Salim
100696781
BMS 673
The Ames test is one of the many biological testing methods available that may be employed
in determining the mutagenesis potential of possible carcinogenic substances. Most of the
other tests rely on directly subjecting mammalian cells to chemical agents undergoing
examination, followed by applying a varied variety of biological readouts. A sister chromatid
exchange (SCE) test, for example, evaluates chromatid crossover between two paired
chromatids produced during the S phase via DNA replication and existing in the paired state
during the late (G2) period of cell development and division cycle. This SCE has been
demonstrated to be triggered by various mutagenic chemicals. Mutagenic substances may
also be capable of causing the production of fractured cell nuclei or micronuclei. The
application of genetics has enabled the selection of mammalian cells lacking thymidine
kinase enzymes due to mutation. The capacity to study the chromosomal sequence of cells in
the metaphase of mitosis under a light microscope allows for detecting chromosomal
abnormalities caused by test substances. Another test measures the extent to which DNA is
labelled in cells in the G1 or G2 stages of the cell cycle; on the grounds that cell DNA
synthesis ordinarily happens in the S stage, such non-S-stage naming, otherwise called
“unscheduled DNA synthesis,” has been demonstrated to be a reliable predictor of genomic
damage done on a cultured cell since this kind of DNA synthesis addresses one key stage in
the process utilized by cells to fix DNA damage. None of these tests has been demonstrated
to be a magnificent indicator of a test substance’s carcinogenicity.
6. In comparison to Aflatoxin B1, is compound X more or less mutagenic? Explain.
Aflatoxin B1 is more mutagenic than compound x. Since both the x and y axes are plotted as
how much compound is necessary to generate a noticeable impact, the mixtures of the most
potent mutagens show up in the lower left of the plot. According to the graph, Aflatoxin B1
appears more on the lower left of the chart than compound x.
7. In comparison to MMS, is compound X more or less carcinogenic? Explain
MMS is less carcinogenic than compound x. Because both the x and y axes are displayed as
the quantity of substance necessary to produce a visible impact. The mixtures that are the
most potent carcinogen show up in the lower left of the plot. Compound x appears more on
the lower left of the graph than MMS.
8. In mouse model experiments, compound X results in the formation of adenomas in
skin epithelial tissue within four months of exposure. How likely is the tumour to
metastasize to other tissues? Explain
Adenomas tumors are unlikely to metastasize
Adenomas tumors are benign (non-cancerous) in nature. As a result, they tend to grow big,
but they will not invade nearby tissues or metastasis to different body areas.
Sabri Abu Salim
100696781
BMS 673
9.1. Outline the architecture of the normal (wild-type) EGF receptor (EGFR) that
allows it to respond to extracellular signals.
A wide range of protein couriers engages with a diverse group of cell surface receptors that
transmit signals across the plasma layer into the cytoplasm. A complex network of signaltransducing proteins processes these signals in the nucleus, funneling them into the nucleus
and eventually elicits a range of biological reactions. Many components of this circuitry, both
on the cell surface and within the cell, have a role in cancer pathogenesis. This cartoon
focuses on a tiny fraction of the receptors found on the surfaces of mammalian cells—the
EGF receptor and its relatives.
9.2. How does the extracellular signal result in a change in cellular behavior?
When they bind an extracellular signal molecule (a ligand), they activate and release a
cascade of intracellular signals that change the cell’s activity.
10.1. What is the catalytic activity of EGFR?
The EGFR is a tyrosine kinase receptor important in cell proliferation and is frequently
misregulated in cancer.
10.2. How can the structural similarity between EGFR and src protein be used to
understand the catalytic activity of src protein?
Comparing the amino acid sequences of the EGF-R and Src cytoplasmic domains found
regions of sequence similarity, indicating that the EGF-R, like Src, generates signals by
acting as a tyrosine kinase. Due to their structural similarities, the activation of EGFR
increases the catalytic activity of src protein.
11.1. Outline three different mechanisms by which normal wild-type EGFR can be
converted into an oncogene.
Growth factor autocrine/paracrine loops
EGFR signaling is promoted by activating mutations.
11.2. Explain what the change is and how it brings about oncogenic function.
Growth factor autocrine/paracrine loops
A paracrine interaction occurs when proteins created by one cell spread out across small
distances to effect change in nearby cells. Paracrine factors, or growth and differentiation
factors (GDFs), are the names given to diffusible proteins.
EGFR signaling is promoted by activating mutations.
Kinase domain mutations in EGFR are referred to as ‘activating mutations’ because they
cause TK activity to be activated without the presence of a ligand. A second mutation in
some malignancies can render partly activated mutant EGFRs entirely ligand-independent
and hence constitutively active.
Sabri Abu Salim
100696781
BMS 673
12. How do gene fusion events drive oncogene formation in the case of receptors such as
FGF-R3?
The fusion protein FGFR3-TACC3 is found at mitotic spindle poles, causing Abnormal
mitosis and chromosomal segregation, which supplement glioblastoma aneuploidy.
13. Explain the difference between active and inactive forms of wild-type Ras protein.
Activate Ras proteins bind to GTP molecule while inactive Ras protein binds to GDP
molecule.
14. Explain the role of GAPs and GEFs in converting Ras between active and inactive
forms.
Ras proteins usually reside in a state of balance between inactive (Ras-GDP) and active (RasGTP). Ras activates many signaling cascades downstream; however, differences in Ras
localization to plasma membrane subdomains and new functions for some scaffold proteins
lead to signaling specificities of the various Ras proteins. Even though Ras proteins
incorporate GTPase and GDP/GTP exchange abilities, they are unequipped for clarifying the
speedy and transient GDP/GTP cycling seen during mitogenic activation. Ras functions
consequently request the presence of administrative proteins that control the pace of
GDP/GTP cycling. GTPase initiating proteins (Ras-GAPs) stimulate the hydrolysis of bound
GTP to GDP, though Ras-GEFs advance the replacement of bound GDP with GTP.
15. An analysis of the frequency of ras oncogenic mutations seen in >9000 tumors is
shown on the left. Explain the results seen from this analysis
The sequencing of almost 10,000 K-ras oncogenes in human tumor uncovered that, rather
than happening randomly all through the genes 189 codon frame for reading, changes
influence only the nucleotides forming codon 12; changes influence codon 13 just around
12% of the time, and codon 61 just every so often. Since all of the K-ras reading frame
codons are probably going to be changed at tantamount rates during cancer development, this
recommends that a substantial specific tension favors the outgrowth of cells with changes
influencing codons 12, 13, and 61. However, mutations affecting different codons in the
reading frame have no impact on cell phenotype or are effectively disadvantageous for the
cells bearing them.
16. What is the molecular cause of the oncogenic activity of the ras mutations seen in the
figure?
Tumors are related to precise Ras gene mutations at codons 12, 13, 59, and 61. These
mutations encourage constitutive Ras activation, Thus implying that the gene is always
“active,” and the protein is overproduced. Additionally, the mutation enhances GTP binding,
resulting in overactivity.
Sabri Abu Salim
100696781
BMS 673
17.1. In response to the presence of mitogens in the environment, cells transition across
the Restriction, R-point in late G1. What is the role of the R-point?
To ensure that the cell is ready to divide, it checks to see whether it has enough proteins and
organelles to go through mitosis, and, most crucially for our purposes, it examines the DNA
to ensure that it is not damaged.
17.2. Using self-drawn images, explain the molecular cascade that transitions across Rpoint due to the presence of mitogens.
17.3. Make sure to elaborate on the role of CDK4/6-Cyclin D, CDK2-CyclinE, INK4s,
CIKs, pRb, E2F in this cascade. 10 points
A molecular “gate” serves as the restriction point for cell-cycle progression, where gene
expression regulation is necessary. The passage is made up of proteins that are linked to the
Rb susceptibility protein and the E2F family of critical transcription factors. In a nutshellE2F
is a central translational regulator that activates many genes whose products are responsible
for Cell-cycle advancement and DNA replication. By attaching to E2F, Rb controls the cell
cycle and changes it into a cell-cycle gene repressor. The cell is considered constrained at the
R-point when Rb binds to E2F. Cdk stimulation, followed by Rb phosphorylation, is required
to break free from this halt. This results in the release of E2F, which then promotes cell-cycle
advancement. Another method for bypassing the R-point gate relies on a potent regulator
known as Myc.
18.1. Why is Rb considered a tumor suppressor?
When a cell’s DNA is broken, Rb inhibits it from replicating by blocking the cell cycle from
progressing from G1 to the Synthesis phase.
Sabri Abu Salim
100696781
BMS 673
18.2. Explain the different ways in which loss of heterozygosity occurs at the Rb locus to
result in cancer formation.
loss of heterozygosity due to mitotic recombination
When chromosomes do not form correctly during cell division, a genetic code might result in
a typo or defect, possibly allowing cells to multiply indefinitely – a hallmark of cancer.
loss of heterozygosity due to missegregation during mitosis
Gene mutation can cause cancerous tumors by accelerating the pace of cell division or by
impeding typical framework reactions, like, cell cycle capture or customized cell death.
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