BIOL 266 – CELL BIOLOGY

Lecture 17:
Cancer and Apoptosis
 The fundamental abnormality resulting in the development of
cancer is the continual unregulated proliferation of cancer cells

Rather than responding appropriately to the signals that control

normal cell growth, division, death and survival, cancer cells grow
and divide in an uncontrolled manner, invading normal tissues and
organs and eventually spreading throughout the body

normal cells cancer cells

epithelial tissue
Tumor – any group of
cells that grow and divide
in an uncontrolled
manner
blood vessel

invasion – distributing into

surrounding normal tissues

spreading throughout the body (the process called metastasis)

 DNA damage arrests cell cycle progression at a checkpoint in G1
low concentration high concentration
of inactive p53 of active p53

DNA
p21 gene

p21 mRNA
p21 protein
(S phase Cdk
cyclin A inhibitor protein) cyclin A
S phase Cdk S phase Cdk
P P
G1 checkpoint active inactive
STOP
STOP
G1 S G2 M G1 checkpoint:
DNA is damaged!!!
The p21 protein binds to the “cyclin A - S phase Cdk” complex and inactivates it,
so that the cell cycle arrests in G1 phase
The arrest of the cell cycle in G1 allows the cell time to repair the damaged DNA
before replicating it
 The gene encoding p53 is frequently mutated in human cancers

DNA
p21 gene

cyclin A
S phase Cdk
G1 checkpoint
P
active
No STOP! STOP

G1 S G2 M

The unrestrained replication of damaged DNA leads to a high rate of

mutation and the production of cells that tend to become cancerous
Mutations in the p53 gene that permit cell with damaged DNA to divide
play an important part in the development of most human cancers
 Three main groups of cancers:

Carcinomas Sarcomas Leukemias

Cancers that arise Cancers that arise & Lymphomas
from epithelial cells from connective-
tissue cells (muscle, Cancers that arise from
bone) the blood-forming cells
and from cells of the
90% of human
immune system,
cancers
2% of human respectively
cancers

8% of human cancers
 The development of cancer is a multistep process involving mutation and selection
of cells with progressively increased capacity for proliferation (division), survival,
invasion and metastasis

normal cells

epithelial tissue

(1) Tumor initiation

a single cell carrying

an initial mutation
Step 1:
A mutation in one cell slightly increases its capacity for proliferation
The mutated cell is able to divide when it should not
The development of cancer is a multistep process involving mutation and selection
of cells with progressively increased capacity for proliferation (division), survival,
invasion and metastasis

(2) Proliferation of
the initial tumor cell

initial tumor cell population

Step 2:
Proliferation of the initial tumor cell gives rise to an initial tumor cell population
The development of cancer is a multistep process involving mutation and selection
of cells with progressively increased capacity for proliferation (division), survival,
invasion and metastasis

(3) A second mutation

increases growth potential

variant tumor cell with

Step 3: increased growth potential

One of the tumor progeny cells undergoes a second mutation that confers a
selective advantage to the cell, such as more rapid growth, and allows the cell to
outgrow the others
The development of cancer is a multistep process involving mutation and selection
of cells with progressively increased capacity for proliferation (division), survival,
invasion and metastasis

(4) Selection for rapid growth

still more rapidly growing

Step 4: variant cell

The tumor cell carrying the second mutation outgrows the other tumor cells
One of the tumor cells carrying the second mutation undergoes a third mutation
that further increases its growth rate and allows the cell to outgrow the other
tumor cells
The development of cancer is a multistep process involving mutation and selection
of cells with progressively increased capacity for proliferation (division), survival,
invasion and metastasis

(5) Further selection for rapid growth

Rapidly growing
variant tumor
cell population

Step 5:
The tumor cell carrying the third mutation outgrows the other tumor cells and
gives rise to a rapidly growing variant tumor cell population
Each cell in this population has all three mutations
 The development of cancer is a multistep process involving mutation and selection
of cells with progressively increased capacity for proliferation (division), survival,
invasion and metastasis

(6) Selection for increased capacity

for invasion and metastasis

blood vessel

invasion metastasis

Step 6:
An additional mutation in one of the rapidly growing tumor cells allows its progeny to escape
into the blood and establish daughter cancer cells at other sites
Since decades are required for these multiple mutations to occur, the exponential increase in
cancer incidence with age is observed
 Causes of cancer
Substances that cause cancer are called carcinogens
Two groups of carcinogens:
(1) initiating agents – induce mutations in key target genes, called proto-oncogenes,
thereby initiating cancer development
- solar ultraviolet radiation (the major cause of skin cancers)
- carcinogenic chemicals in tobacco smoke (the major identified causes of lung
cancers)
- aflatoxin (a potent liver carcinogen produced by some molds that contaminate
improperly stored supplies of peanuts)

(2) tumor promoters – stimulate cell proliferation (division), rather then induce
mutations in proto-oncogenes
- phorbol esters (stimulate early stages of tumor development by activating protein
kinase C)
- estrogens, a group of steroid hormones (long-term estrogen replacement therapy
results in the development of some human cancers)
 Properties of cancer cells
1st property: The proliferation of cancer cells in cell
culture is not sensitive to density-dependent inhibition
Normal cells proliferate in culture until they rich a finite cell density, at which
point they become arrested in the G0 stage of the cell cycle
Cancer cells continue to proliferate independent of their density in culture

Normal cells cells

cell cells stop

proliferation no serum growth proliferating
serum growth factors factors

Cancer cells cells

cell cell
proliferation no serum growth proliferation
serum growth factors factors continues
 Properties of cancer cells
1st property: The proliferation of cancer cells in cell
culture is not sensitive to density-dependent inhibition

If deprived of growth factors, normal cells are arrested at a G1 checkpoint called

the restriction point
When arrested at the restriction point, normal cells enter a specific stage of the cell
cycle called G0, in which they dismantle their cell-cycle control system and
withdraw from the cell cycle

cyclin B
cyclin E P
cyclin A
G1 phase Cdk M phase Cdk
S phase Cdk
P P

G1 S G2 M

STOP

G0 restriction point

Growth factors are not available!!!

 Properties of cancer cells
1st property: The proliferation of cancer cells in cell
culture is not sensitive to density-dependent inhibition

If deprived of growth factors, cancer cells are NOT arrested at a G1 checkpoint

called the restriction point
If deprived of growth factors, cancer cells do not dismantle their cell-cycle control
system and do not withdraw from the cell cycle

cyclin E cyclin B
P
cyclin A
G1 phase Cdk M phase Cdk
S phase Cdk
P P

G1 S G2 M

Growth factors are not available? So what!!!

 Properties of cancer cells
2nd property: Some cancer cells produce growth factors that stimulate their
own proliferation
Such abnormal production of a growth factor by a target cell leads to
continuous autostimulation of cell division (autocrine growth stimulation)

cancer growth factor

normal cell cell
growth factor receptor stimulation

cancer cancer
cell cell

cancer cancer cancer cancer

cell cell cell cell
 Properties of cancer cells
3rd property: The reduced growth factor dependence of many cancer cells results
from mutations in genes encoding proteins (e.g., Ras proteins or protein kinases)
that function in the signal transduction pathways controlling normal cell
proliferation
Normal version of any gene that encodes a protein which, only when mutated, able
to contribute to the development of cancer is called proto-oncogene
Mutant version of a proto-oncogene, whose encoded oncoprotein induces cancer, is
called oncogene
Normal Ras gene (proto-oncogene) Mutant RasD gene (oncogene)

Ras mRNA RasD mRNA

GDP GTP GTP

Normal Ras protein
Normal Ras protein is present in its active,
the GTP-bound, state only when the “Growth
factor receptor – Ras – Raf – MAP kinase”
signaling pathway is activated by a growth
factor
Mutant RasD protein (oncoprotein)
Mutant RasD protein is always present in its
active, the GTP-bound, state, even in the absence
of the growth factors normally required to
activate the “Growth factor receptor – Ras –
Raf – MAP kinase” signaling pathway
 Properties of cancer cells
No Growth
Mutant RasD protein is always present in its active, the GTP-bound, state, even in the
factor!!!
absence of the growth factors normally required to activate the “Growth factor
receptor – Ras – Raf – MAP kinase” signaling pathway that in turn stimulates
Receptor passage through the restriction point of the cell cycle
tyrosine
kinase TCF
P P ATP ADP SRF
P P
RasD active pp90
P
ATP P
Raf
SRE
ADP ADP ~ 100 early-response genes
active
MAP
P early-response
kinase P ATP
mRNAs
active MAP kinase

nucleus
Stimulate passage through the
restriction point, permitting
the G1 S transition
~ 100 early-response proteins
delayed-response genes
encoding Cdks and cyclins
 Properties of cancer cells
4th property: cancer cells secrete proteases that digest extracellular matrix
components, thereby allowing the cancer cells to digest and penetrate through
basal laminae and to invade underlying connective tissue and other adjacent
normal tissues secreted
normal cells cancer cells proteases

epithelial tissue
basal laminae

connective
tissue

epithelial tissue
basal laminae
invasion
connective
tissue
 Properties of cancer cells

5th property: cancer cells fail to undergo programmed cell death, or apoptosis,
and therefore exhibit increased life spans compared to their normal
counterparts
A failure of tumor cells to undergo apoptosis is important not only in primary
tumor development but also in the survival and growth of metastatic cancer
cells in abnormal tissue sites
 Programmed cell death (apoptosis)

Programmed cell death (also called apoptosis) is a normal physiological form of

cell death that plays a key role in the maintenance of adult tissues
In adults, apoptosis is responsible for:
(1) eliminating virus-infected cells, thereby preventing the production of new
virus particles and limiting spread of the virus through the host organism

virus-infected cell normal cell

(2) eliminating cells carrying damaged DNA, thereby eliminating cells with
mutations that might lead to the development of cancer

normal cells

cell with
damaged DNA cancer cell
 Morphologic changes in dying (apoptotic) cells

cell
nucleus

chromatin
Nucleosome
Step 1: DNA fragmentation

DNA cleavage

Step 1:
Chromosomal DNA is fragmented as a result of cleavage between
nucleosomes
 Morphologic changes in dying (apoptotic) cells

Step 2: chromatin condensation

and fragmentation of nucleus

Step 2:
The chromatin condenses and the nucleus then breaks up into
small pieces
 Morphologic changes in dying (apoptotic) cells

Step 3: fragmentation of the cell

apoptotic bodies

Step 3:
The cell itself shrinks and breaks up into membrane-enclosed
fragments called apoptotic bodies
 Morphologic changes in dying (apoptotic) cells

apoptotic bodies

Step 4: phagocytosis

Step 4:
Apoptotic bodies are phagocytosed by both macrophages
(specialized phagocytic cells) and neighboring cells, before there
is any leakage of cell contents into the extracellular space
 Mechanism of apoptosis
Survival factor receptor (receptor tyrosine kinase)

Plasma membrane

Cytosol Cascade of suicide

Bad proteases
Cleavage of
Suicide protease
Caspase 9
key proteins
Procaspase 9
(1) leading to
Apaf 1 Cytochrome c ions cell death
Bcl-XL

Bad
Bcl-2

Bax
Bax
Outer mitochondrial membrane

Cytochrome c

Step 1:
In the absence of a survival factor, which suppresses apoptosis, a soluble pro-apoptotic
protein, Bad, binds to the anti-apoptotic proteins Bcl-2 and Bcl-XL, which are inserted
into the outer mitochondrial membrane
 Mechanism of apoptosis
Survival factor receptor (receptor tyrosine kinase)

Plasma membrane

Cytosol Cascade of suicide

Bad proteases
Cleavage of
Suicide protease
Caspase 9
key proteins
Procaspase 9
leading to
Apaf 1 Cytochrome c ions cell death
Bcl-XL

Bad
Bcl-2

Bax
Bax
Outer mitochondrial membrane

Cytochrome c (2)

Step 2:
Bad binding prevents the anti-apoptotic proteins Bcl-2 and Bcl-XL from interacting
with Bax, a mitochondrial membrane-bound pro-apoptotic protein
 Mechanism of apoptosis
Survival factor receptor (receptor tyrosine kinase)

Plasma membrane

Cytosol Cascade of suicide

Bad proteases
Cleavage of
Suicide protease
Caspase 9
key proteins
Procaspase 9
leading to
Apaf 1 Cytochrome c ions cell death
Bcl-XL

Bad
Bcl-2

(3)

Bax
Bax
Outer mitochondrial membrane

Cytochrome c

Step 3:
As a consequence, Bax forms homo-dimeric channels in the outer mitochondrial
membrane
These channels mediate ion flux
 Mechanism of apoptosis
Survival factor receptor (receptor tyrosine kinase)

Plasma membrane

Cytosol Cascade of suicide

Bad proteases
Cleavage of
Suicide protease
Caspase 9
key proteins
Procaspase 9
leading to
Apaf 1 Cytochrome c ions cell death
Bcl-XL

Bad (4)
Bcl-2

Bax
Bax
Outer mitochondrial membrane

Cytochrome c

Step 4:
The ion flux leads to the release of cytochrome c from the space between the inner
and outer mitochondrial membrane to the cytosol
 Mechanism of apoptosis
Survival factor receptor (receptor tyrosine kinase)

Plasma membrane

Cytosol Cascade of suicide

Bad proteases
Cleavage of
Suicide protease
(5)
Caspase 9
key proteins
Procaspase 9
leading to
Apaf 1 Cytochrome c ions cell death
Bcl-XL

Bad
Bcl-2

Bax
Bax
Outer mitochondrial membrane

Cytochrome c

Step 5:
In the cytosol, cytochrome c binds to the adapter protein Apaf 1, which in turn leads
to autocleavage and activation of a suicide protease called caspase 9
 Mechanism of apoptosis
Survival factor receptor (receptor tyrosine kinase)

Plasma membrane

Cytosol Cascade of suicide

Bad proteases
Cleavage of
Suicide protease
Caspase 9
key proteins
Procaspase 9
leading to
Apaf 1 Cytochrome c (6) cell death
ions
Bcl-XL

Bad
Bcl-2

Bax
Bax
Outer mitochondrial membrane

Cytochrome c

Step 6:
The activated suicide protease, caspase 9, cleaves and activates other members of the
family of suicide proteases, resulting in an amplifying proteolytic cascade
The activated caspases then cleave key proteins in the cell, killing it quickly
 Key targets of the caspases (suicide proteases):

(1) an inhibitor of a DNAse which, when activated, is responsible for

fragmentation of nuclear DNA

(2) nuclear lamins, whose proteolytic cleavage leads to fragmentation

of the nucleus

(3) cytoskeletal proteins, whose proteolytic cleavage leads to

disruption
of the cytoskeleton and cell fragmentation
survival Mechanism of cell survival
factor
Survival factor receptor (receptor tyrosine kinase)

Plasma membrane

(1) Cytosol Cascade of suicide

Bad proteases
Cleavage of
Suicide protease
P Caspase 9
key proteins
Procaspase 9
leading to
Apaf 1 Cytochrome c cell death
Bcl-XL
Bcl-2

Bax
Bax
Outer mitochondrial membrane

Cytochrome c
Step 1:
In the presence of a survival factor, the survival factor binds to its receptor, a receptor tyrosine
kinase
The survival factor-associated receptor activates the downstream “PIP3 – PDK kinase – Akt
kinase” signaling cascade
The activated Akt kinase phosphorylates the cytosolic pro-apoptotic protein Bad
survival Mechanism of cell survival
factor
Survival factor receptor (receptor tyrosine kinase)

Plasma membrane

Cytosol Cascade of suicide

Bad proteases
Cleavage of
Suicide protease
P Caspase 9
key proteins
Procaspase 9
leading to
Apaf 1 Cytochrome c cell death
(2)
Bcl-XL
Bcl-2

Bax
Bax
Outer mitochondrial membrane

Cytochrome c
Step 2:
Phosphorylated Bad is unable to bind to the anti-apoptotic proteins Bcl-2 and Bcl-XL
With Bad located in the cytosol, Bcl-2 and Bcl-XL inhibit the activity of Bax, thereby
preventing the release of cytochrome c and activation of the cascade of suicide proteases
 In the absence of a survival factor, normal cells undergo apoptosis
Survival factor receptor (receptor tyrosine kinase)

Plasma membrane

Cytosol Cascade of suicide

Bad proteases Cleavage of
Suicide protease
key proteins
Procaspase 9 Caspase 9
leading to cell
Apaf 1 Cytochrome c death
ions
Bcl-XL

Bad
Bcl-2

Bax
Bax
Outer mitochondrial membrane

Cytochrome c

Normal cells:
In the absence of a survival factor, which suppresses apoptosis, the anti-apoptotic
proteins Bcl-2 and Bcl-XL are unable to interact with Bax, thereby promoting the
release of cytochrome c and activation of the cascade of suicide proteases
survival In the presence of a survival factor, normal cells survive
factor
Survival factor receptor (receptor tyrosine kinase)

Plasma membrane

Cytosol Cascade of suicide

Bad proteases Cleavage of
Suicide protease
key proteins
P Procaspase 9 Caspase 9
leading to cell
Apaf 1 Cytochrome c death
Bcl-XL
Bcl-2

Bax
Bax
Outer mitochondrial membrane

Cytochrome c

Normal cells:
In the presence of a survival factor, which suppresses apoptosis, the anti-apoptotic
proteins Bcl-2 and Bcl-XL inhibit the activity of Bax, thereby preventing the
release of cytochrome c and activation of the cascade of suicide proteases
 Cancer cells carrying the Bcl-20 mutation
survive even in the absence of a survival factor
Survival factor receptor (receptor tyrosine kinase)

Plasma membrane

Cytosol Cascade of suicide

Bad proteases Cleavage of
Suicide protease
key proteins
Procaspase 9 Caspase 9
leading to cell
Apaf 1 Cytochrome c death
Bcl-XL

Bad
Bcl-20

Bax
Bax
Outer mitochondrial membrane

Oncoprotein!
Cytochrome c

Cancer cells:
Even in the absence of a survival factor, the oncoprotein Bcl-20 inhibits the activity of
Bax, thereby preventing the release of cytochrome c and activation of the cascade of
suicide proteases